Twisted gastrulation polypeptides and uses thereof

ABSTRACT

In certain aspects, the present invention provides compositions and methods for altering iron metabolism to increase red blood cell and/or hemoglobin levels in vertebrates, including rodents and primates, and particularly in humans.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/395,088, filed Sep. 15, 2016, which isherein incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 8, 2017, isnamed APH-00125_SL.txt and is 27,134 bytes in size.

BACKGROUND OF THE INVENTION

The mature red blood cell (RBC), or erythrocyte, is responsible foroxygen transport in the circulatory systems of vertebrates. Red bloodcells contain high concentrations of hemoglobin, a protein that bindsoxygen in the lungs at relatively high partial pressure of oxygen (pO₂)and delivers oxygen to areas of the body with a relatively low pO₂.

Mature red blood cells are produced from pluripotent hematopoietic stemcells in a process termed erythropoiesis. Postnatal erythropoiesisoccurs primarily in the bone marrow and in the red pulp of the spleen.The coordinated action of various signaling pathways controls thebalance of cell proliferation, differentiation, survival and death.Under normal conditions, red blood cells are produced at a rate thatmaintains a constant red cell mass in the body, and the rate mayincrease or decrease in response to various stimuli, including increasedor decreased oxygen tension or tissue demand. The process oferythropoiesis begins with the formation of lineage committed precursorcells and proceeds through a series of distinct precursor cell types.The final stages of erythropoiesis occur as reticulocytes are releasedinto the bloodstream and lose their mitochondria and ribosomes whileassuming the morphology of mature red blood cell. An elevated level ofreticulocytes, or an elevated reticulocyte:erythrocyte ratio, in theblood is indicative of increased red blood cell production rates.

Erythropoietin (EPO) is widely recognized as the most significantpositive regulator of postnatal erythropoiesis in vertebrates. EPOregulates the compensatory erythropoietic response to reduced tissueoxygen tension (hypoxia) and low red blood cell levels or low hemoglobinlevels. In humans, elevated EPO levels promote red blood cell formationby stimulating the generation of erythroid progenitors in the bonemarrow and spleen. In the mouse, EPO enhances erythropoiesis primarilyin the spleen.

Effects of EPO are mediated by a cell-surface receptor belonging to thecytokine receptor superfamily. The human EPO receptor gene encodes a 483amino-acid transmembrane protein, whereas the active EPO receptor isthought to exist as a multimeric complex even in the absence of ligand(See U.S. Pat. No. 6,319,499). The cloned full-length EPO receptorexpressed in mammalian cells binds EPO with an affinity similar to thatof the native receptor on erythroid progenitor cells. Binding of EPO toits receptor causes a conformational change resulting in receptoractivation and biological effects including increased proliferation ofimmature erythroblasts, increased differentiation of immatureerythroblasts, and decreased apoptosis in erythroid progenitor cells(Liboi et al., 1993, Proc Natl Acad Sci USA 90:11351-11355; Koury etal., 1990, Science 248:378-381).

Various forms of recombinant EPO are used by physicians to increase redblood cell levels in a variety of clinical settings, and particularlyfor the treatment of anemia. Anemia is a broadly-defined conditioncharacterized by lower than normal levels of hemoglobin or red bloodcells in the blood. In some instances, anemia is caused by a primarydisorder in the production or survival of red blood cells. Morecommonly, anemia is secondary to diseases of other systems (Weatherall &Provan, 2000, Lancet 355, 1169-1175). Anemia may result from a reducedrate of production or increased rate of destruction of red blood cellsor by loss of red blood cells due to bleeding. Anemia may result from avariety of disorders that include, for example, chronic renal failure,chemotherapy treatment, myelodysplastic syndrome, rheumatoid arthritis,and bone marrow transplantation.

Treatment with EPO typically causes a rise in hemoglobins by about 1-3g/dL in healthy humans over a period of weeks. When administered toanemic individuals, this treatment regimen often provides substantialincreases in hemoglobin and red blood cell levels and leads toimprovements in quality of life and prolonged survival. EPO is notuniformly effective, and many individuals are refractory to even highdoses (Horl et al., 2000, Nephrol Dial Transplant 15, 43-50). Over 50%of patients with cancer have an inadequate response to EPO,approximately 10% with end-stage renal disease are hyporesponsive(Glaspy et al., 1997, J Clin Oncol 15, 1218-1234; Demetri et al., 1998,J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplasticsyndrome respond favorably (Estey, 2003, Curr Opin Hematol 10, 60-67).Several factors, including inflammation, iron and vitamin deficiency,inadequate dialysis, aluminum toxicity, and hyperparathyroidism maypredict a poor therapeutic response. The molecular mechanisms ofresistance to EPO are as yet unclear. Recent evidence suggests thathigher doses of EPO may be associated with an increased risk ofcardiovascular morbidity, tumor growth, and mortality in some patientpopulations (Krapf et al., 2009, Clin J Am Soc Nephrol 4:470-480;Glaspy, 2009, Annu Rev Med 60:181-192). It has therefore beenrecommended that EPO-based therapeutic compounds(erythropoietin-stimulating agents, ESAs) be administered at the lowestdose sufficient to avoid the need for red blood cell transfusions(Jelkmann et al., 2008, Crit Rev Oncol. Hematol 67:39-61).

Thus, there is a need for alternative methods for increasing numbers ofred blood cells and levels of iron available for erythropoiesis in thecontext of anemia of inflammation.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides novel Twistedgastrulation (TWSG) polypeptides comprising, e.g., an isolated TWSGpolypeptide, a soluble TWSG polypeptide, or an at least partly purifiedTWSG polypeptide. In some embodiments, the present disclosure providesan isolated, soluble, and at least partly purified TWSG polypeptide.

In some embodiments, the present disclosure provides a polypeptidecomprising a TWSG polypeptide and another polypeptide, e.g., apolypeptide heterologous to the TWSG polypeptide. In some embodiments,the heterologous polypeptide is a fragment crystallizable region (Fe)polypeptide. The polypeptide may be, for example, a fusion proteincomprising a TWSG polypeptide and an Fc polypeptide.

In some embodiments, the TWSG polypeptide is a human or non-humanvertebrate TWSG polypeptide, or a chimeric TWSG polypeptide comprisingat least part of a human TWSG polypeptide and at least part of anon-human vertebrate TWSG polypeptide. Representative non-humanvertebrates include, for example, non-human mammals, such as a non-humanprimate, a mouse, a rat, a rabbit, a dog, a cat, a pig, a sheep, a cow,a horse, a donkey, a camel, etc., and non-human non-mammal vertebrates(e.g., chicken, snake, etc.). Non-human primates include, for example, amonkey, a chimpanzee, a gibbon, a macaque, a gorilla, an orangutan, etc.

In certain embodiments, the TWSG polypeptide of the present disclosureis encodable by a polynucleotide comprising a nucleic acid sequence atleast about 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100% identical to the sequence of SEQ ID NO: 2.

In some embodiments, the TWSG polypeptide is encodable by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 2. Insome embodiments, the TWSG polypeptide is encodable by a polynucleotidethat hybridizes, e.g., under highly stringent conditions, to a nucleicacid sequence that is complementary to the sequence of SEQ ID NO: 2.Such highly stringent conditions may comprise hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. to about 65° C., followedby at least one wash in about 0.2×SSC to about 2.0×SSC at about 50° C.to about 65° C. In some embodiments, such highly stringent conditionsmay comprise hybridization in 6× sodium chloride/sodium citrate (SSC) atabout 65° C., followed by a wash in 0.2×SSC at about 65° C.

In certain embodiments, the TWSG polypeptide of the present disclosurecomprises an amino acid sequence at least about 60, 65, 70, 75, 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to thesequence of SEQ ID NO: 8. In some embodiments, the TWSG polypeptidecomprises an amino acid sequence at least 80% identical to the sequenceof SEQ ID NO: 8. In some embodiments, the TWSG polypeptide comprises anamino acid sequence at least 85% identical to the sequence of SEQ ID NO:8. In some embodiments, the TWSG polypeptide comprises an amino acidsequence at least 90% identical to the sequence of SEQ ID NO: 8. In someembodiments, the TWSG polypeptide comprises an amino acid sequence atleast 95% identical to the sequence of SEQ ID NO: 8. In someembodiments, the TWSG polypeptide comprises an amino acid sequence atleast 99% identical to the sequence of SEQ ID NO: 8. In someembodiments, the TWSG polypeptide comprises an amino acid sequence ofSEQ ID NO: 8.

In some embodiments, the TWSG polypeptide of the present disclosure is awild-type or naturally occurring TWSG polypeptide. In other embodiments,the TWSG polypeptide comprises at least one amino acid substitutionrelative to a wild-type or naturally occurring sequence. Such amino acidsubstitution may be, for example, in a potential glycosylation site inorder to, for example, remove a potential glycosylation site. Such aminoacid substitution may be, for example, in a site potentially affectingthe biological function and/or interaction affinity of the TWSGpolypeptide.

In some embodiments, the TWSG polypeptide comprises a signal sequence.Such signal sequence may be at, e.g., the N-terminus of the TWSGpolypeptide. In other embodiments, the TWSG polypeptide does notcomprise a signal sequence.

In some embodiments, the Fc polypeptide is a human or non-humanvertebrate Fc polypeptide. Such non-human vertebrate may be, forexample, a non-human mammal, including, for example, a non-humanprimate. The Fc polypeptide may be derived from an IgG protein, such asIgG1, IgG2, IgG3, IgG4, or a chimeric IgG subclass (e.g., IgG2/G4). Insome embodiments, the Fc polypeptide is derived from IgG1.

In some embodiments, the Fc polypeptide is encodable by a polynucleotidethat hybridizes, e.g., under highly stringent conditions, to a nucleicacid sequence that is complementary to the sequence of a polynucleotideencoding a polypeptide comprising an amino acid sequence of SEQ ID NO:3, 4, 5, 6, or 7. Such highly stringent conditions may comprisehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C.to about 65° C., followed by at least one wash in about 0.2×SSC to about2.0×SSC at about 50° C. to about 65° C. In some embodiments, such highlystringent conditions may comprise hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 65° C., followed by a wash in0.2×SSC at about 65° C.

The Fc polypeptide may comprise an amino acid sequence at least about60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7. In someembodiments, the Fc polypeptide comprises an amino acid sequence atleast 80% identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7. Insome embodiments, the Fc polypeptide comprises an amino acid sequence atleast 85% identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7. Insome embodiments, the Fc polypeptide comprises an amino acid sequence atleast 90% identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7. Insome embodiments, the Fc polypeptide comprises an amino acid sequence atleast 95% identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7. Insome embodiments, the Fe polypeptide comprises an amino acid sequence atleast 99% identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7. Insome embodiments, the Fc polypeptide comprises an amino acid sequence ofSEQ ID NO: 3, 4, 5, 6, or 7.

In some embodiments, the Fc polypeptide is a wild-type or naturallyoccurring Fe polypeptide. In other embodiments, the Fe polypeptidecomprises at least one amino acid substitution relative to a wild-typeor naturally occurring sequence. Such amino acid substitution may be,for example, in a potential glycosylation site in order to, for example,remove a potential glycosylation site. Such amino acid substitution maybe, for example, in a site potentially affecting the biological functionand/or interaction affinity of the Fc polypeptide.

The polypeptide of the present disclosure may be a fusion proteincomprising a TWSG polypeptide and a Fe polypeptide. In such embodiments,the C-terminus of TWSG polypeptide may be fused to the N-terminus of theFc polypeptide. In other such embodiments, the C-terminus of the Fepolypeptide may be fused to the N-terminus of the TWSG polypeptide. Suchfusion proteins, regardless of the order of these elements, arecollectively referred to herein as “TWSG-Fc” in the present disclosure.

In some embodiments, a fusion protein as disclosed herein may furthercomprise a linker sequence between the TWSG polypeptide and the Fcpolypeptide. Such linker sequence may be of any length permitted orpreferred for general fusion proteins or for the fusion protein ofTWSG-Fc. For example, the linker sequence may comprise of a structure ofX-(G)n-Y, wherein X is absent or at least one amino acid residue,preferably T or S, wherein n is an integer having a value of at least 1,preferably 3 or 4; and wherein Y is absent or at least one amino acidresidue, preferably S. In some embodiments, the linker sequencecomprises TGGG (SEQ ID NO: 16), SGGG (SEQ ID NO: 17), TGGGG (SEQ ID NO:18), SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20), GGGG (SEQ ID NO: 21),or GGG, preferably TGGG (SEQ ID NO: 16).

In certain aspects, the present disclosure provides a TWSG-Fc fusionprotein comprising a polypeptide encodable by a polynucleotidecomprising a nucleic acid sequence at least about 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to thesequence of SEQ ID NO: 14. In some embodiments, the polypeptide isencodable by a polynucleotide comprising a nucleic acid sequence atleast 80% identical to the sequence of SEQ ID NO: 14. In someembodiments, the polypeptide is encodable by a polynucleotide comprisinga nucleic acid sequence at least 85% identical to the sequence of SEQ IDNO: 14. In some embodiments, the polypeptide is encodable by apolynucleotide comprising a nucleic acid sequence at least 90% identicalto the sequence of SEQ ID NO: 14. In some embodiments, the polypeptideis encodable by a polynucleotide comprising a nucleic acid sequence atleast 95% identical to the sequence of SEQ ID NO: 14. In someembodiments, the polypeptide is encodable by a polynucleotide comprisinga nucleic acid sequence at least 99% identical to the sequence of SEQ IDNO: 14. In some embodiments, the polypeptide is encodable by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 14.

In some embodiments, the polypeptide is encodable by a polynucleotidethat hybridizes, e.g., under highly stringent conditions to a nucleicacid sequence that is complementary to the sequence of SEQ ID NO: 14.Such highly stringent conditions may comprise hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. to about 65° C., followedby at least one wash in about 0.2×SSC to about 2.0×SSC at about 50° C.to about 65° C. In some embodiments, such highly stringent conditionsmay comprise hybridization in 6× sodium chloride/sodium citrate (SSC) atabout 65° C., followed by a wash in 0.2×SSC at about 65° C.

The present disclosure further provides a TWSG-Fc fusion proteincomprising a polypeptide comprising an amino acid sequence at leastabout 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identical to the sequence of SEQ ID NO: 9. In some embodiments, thepolypeptide comprises an amino acid sequence at least 80% identical tothe sequence of SEQ ID NO: 9. In some embodiments, the polypeptidecomprises an amino acid sequence at least 85% identical to the sequenceof SEQ ID NO: 9. In some embodiments, the polypeptide comprises an aminoacid sequence at least 90% identical to the sequence of SEQ ID NO: 9. Insome embodiments, the polypeptide comprises an amino acid sequence atleast 95% identical to the sequence of SEQ ID NO: 9. In someembodiments, the polypeptide comprises an amino acid sequence at least99% identical to the sequence of SEQ ID NO: 9. In some embodiments, thepolypeptide comprises an amino acid sequence of SEQ ID NO: 9.

In some embodiments, the polypeptide provided in the present disclosureis encodable by a polynucleotide that hybridizes, e.g., under highlystringent conditions to a nucleic acid sequence that is complementary tothe sequence of a polynucleotide encoding a polypeptide comprising anamino acid sequence of SEQ ID NO: 9. Such highly stringent conditionsmay comprise hybridization in 6× sodium chloride/sodium citrate (SSC) atabout 45° C. to about 65° C., followed by at least one wash in about0.2×SSC to about 2.0×SSC at about 50° C. to about 65° C. In someembodiments, such highly stringent conditions may comprise hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 65° C., followed bya wash in 0.2×SSC at about 65° C.

The present disclosure further provides a TWSG-Fc fusion proteincomprising a polypeptide comprising an amino acid sequence at leastabout 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identical to the sequence of SEQ ID NO: 13. In some embodiments,the polypeptide comprises an amino acid sequence at least 80% identicalto the sequence of SEQ ID NO: 13. In some embodiments, the polypeptidecomprises an amino acid sequence at least 85% identical to the sequenceof SEQ ID NO: 13. In some embodiments, the polypeptide comprises anamino acid sequence at least 90% identical to the sequence of SEQ ID NO:13. In some embodiments, the polypeptide comprises an amino acidsequence at least 95% identical to the sequence of SEQ ID NO: 13. Insome embodiments, the polypeptide comprises an amino acid sequence atleast 99% identical to the sequence of SEQ ID NO: 13. In someembodiments, the polypeptide comprises an amino acid sequence of SEQ IDNO: 13.

In some embodiments, the polypeptide provided in the present disclosureis encodable by a polynucleotide that hybridizes, e.g., under highlystringent conditions to a nucleic acid sequence that is complementary tothe sequence of a polynucleotide encoding a polypeptide comprising anamino acid sequence of SEQ ID NO: 13. Such highly stringent conditionsmay comprise hybridization in 6× sodium chloride/sodium citrate (SSC) atabout 45° C. to about 65° C., followed by at least one wash in about0.2×SSC to about 2.0×SSC at about 50° C. to about 65° C. In someembodiments, such highly stringent conditions may comprise hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 65° C., followed bya wash in 0.2×SSC at about 65° C.

In some embodiments, the TWSG-Fc fusion protein or polypeptide of thepresent disclosure comprises wild-type or naturally occurring amino acidsequences for at least one of the TWSG portion and the Fc portion. Inother embodiments, such polypeptide comprises at least one amino acidsubstitution relative to the wild-type or naturally occurring sequences.Such amino acid substitution may be, for example, in a potentialglycosylation site in order to, for example, remove a potentialglycosylation site. Such amino acid substitution may be, for example, ina site potentially affecting the biological function and/or interactionaffinity of the Fe polypeptide. In some embodiments, such polypeptidecomprises at least one amino acid substitution at at least one N-linkedglycosylation site in SEQ ID NO: 9. In other embodiments, suchpolypeptide comprises at least one amino acid substitution at at leastone N-linked glycosylation site in SEQ ID NO: 13.

In certain aspects, the present disclosure provides a TWSG-Fc fusionprotein or polypeptide capable of binding to, preferably inhibiting, atleast one bone morphogenetic proteins (BMP), such as a BMP selected fromBMP2, BMP4, BMP6, BMP7, and BMP9. In some embodiments, the TWSG-Fcfusion protein or polypeptide is capable of binding to, preferablyinhibiting, at least one bone morphogenetic proteins (BMP) with a K_(D)less than 4.5 nM (or preferably, no more than 4.4 nM). In someembodiments, the TWSG-Fc fusion protein or polypeptide is capable ofbinding to, preferably inhibiting, at least one bone morphogeneticproteins (BMP) in a sub-nanomolar affinity. For example, the TWSG-Fcfusion protein or polypeptide may be capable of binding to at least oneof BMPs with a K_(D) no more than 1 nM, 0.5 nM, 0.33 nM, or less. Insome embodiments, the TWSG-Fc fusion protein or polypeptide is capableof binding with a K_(D) no more than 1 nM, 0.5 nM, or 0.33 nM (orpreferably, no more than 0.33, 0.30, or 0.23 nM) to at least one BMPselected from BMP2, BMP4, BMP6, and BMP7. In some embodiments, theTWSG-Fc fusion protein or polypeptide is capable of binding with a K_(D)no more than 1 nM, 0.5 nM, or 0.33 nM (or preferably, no more than 0.33,0.30, or 0.23 nM) to at least two BMPs selected from BMP2, BMP4, BMP6,and BMP7. In some embodiments, the TWSG-Fc fusion protein or polypeptideis capable of binding with a K_(D) no more than 1 nM, 0.5 nM, or 0.33 nM(or preferably, no more than 0.33, 0.30, or 0.23 nM) to at least threeBMPs selected from BMP2, BMP4, BMP6, and BMP7. In some embodiments, theTWSG-Fc fusion protein or polypeptide is capable of binding with a K_(D)no more than 1 nM, 0.5 nM, or 0.33 nM (or preferably, no more than 0.33,0.30, or 0.23 nM) to BMP2, BMP4, BMP6, and BMP7. Such binding betweenthe TWSG-Fc fusion protein or polypeptide and the BMPs may be detectedand/or measured with any suitable technology, such as an in vitrobinding assay detected by surface plasmon resonance.

In some embodiments, the TWSG-Fc fusion protein or polypeptide iscapable of inhibiting at least one of bone morphogenetic proteins(BMPs), such as BMP2, BMP4, BMP6, and BMP7. For example, the TWSG-Fcfusion protein or polypeptide may be capable of inhibiting at least oneof BMPs with an IC₅₀ less than 31 nM.

In some embodiments, the TWSG-Fc fusion protein or polypeptide iscapable of inhibiting at least one of bone morphogenetic proteins (BMPs)with an IC₅₀ at nanomolar level. For example, the TWSG-Fc fusion proteinor polypeptide may be capable of inhibiting at least one of BMPs with anIC₅₀ no more than 5 nM, 4 nM, 3.7 nM, or 3 nM (or preferably, no morethan 3.7, 2.2, or 1.5 nM). In some embodiments, the TWSG-Fc fusionprotein or polypeptide is capable of inhibiting at least one of BMPsselected from BMP4, BMP6, and BMP7 at an IC₅₀ no more than 5 nM or 3.7nM (or preferably, no more than 3.7, 2.2, or 1.5 nM). In someembodiments, the TWSG-Fc fusion protein or polypeptide is capable ofinhibiting at least two BMPs selected from BMP4, BMP6, and BMP7 at anIC₅₀ no more than 5 nM or 3.7 nM (or preferably, no more than 3.7, 2.2,or 1.5 nM). In some embodiments, the TWSG-Fc fusion protein orpolypeptide is capable of inhibiting BMP4, BMP6, and BMP7 at an IC₅₀ nomore than 5 nM or 3.7 nM (or preferably, no more than 3.7, 2.2, or 1.5nM). Such inhibition includes inhibition of at least one aspect of BMPactivity, such as BMP-mediated cell signaling processes. SuchBMP-mediated cell signaling processes may be mediated by at least one ofBMP substrates and/or downstream signaling messengers of BMPs,including, at least, Smad proteins (e.g., Smad 1, Smad 5, etc.). In someembodiments, the TWSG-Fc fusion protein or polypeptide is capable ofinhibiting at least one aspect of BMP-mediated cell signaling processesthrough at least one of Smad1 and Smad 5. Such inhibition may bedetected and/or measured with any suitable technology, such as by acell-based assay, e.g., an in vitro cell-based assay. Such cell-basedassay may utilize at least one BMP response element in, e.g., a pGL3 BREreporter plasmid.

In certain aspects, the present disclosure provides a multimericpolypeptide complex comprising the TWSG-Fc fusion protein or polypeptidedescribed herein. Such complex may comprise the TWSG-Fc fusion proteinor polypeptide and at least one another homologous or heterologouspolypeptide or at least one another molecule of the same TWSG-Fc fusionprotein or polypeptide. Such multimeric polypeptide complex may be, forexample, a dimer, trimer, or an oligomer containing at least 4, 5, 6, 7,8, 9, 10, or more monomer polypeptides. In the multimeric polypeptidecomplex, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of themonomer polypeptides may have the same amino acid sequence (e.g., theTWSG-Fc fusion protein or polypeptide described herein) or have a highsequence homology (e.g., of at least about 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). In someembodiments, the multimeric polypeptide complex is a homodimer (e.g.,comprising two molecules of the TWSG-Fc fusion protein or polypeptide).In other embodiments, the multimeric polypeptide complex is aheterodimer (e.g., comprising TWSG-Fc fusion protein or polypeptide andanother heterologous protein or polypeptide). The monomer polypeptidesin the multimeric polypeptide complex may contact each other through atleast one covalent bond or noncovalent interaction known in the art. Forexample, linkers may be added to such monomer polypeptides to facilitateinteraction to form the multimeric polypeptide complex. In anothernonlimiting example, the Fc portion of the TWSG-Fc fusion protein orpolypeptide may be used to interact with at least one another monomerpolypeptide. In some embodiments, two TWSG-Fc fusion proteins orpolypeptides (with a same Fc portion or different Fe portions comprisingdifferent mutations, substitutions, chemical modifications, etc.) form adimer. Such dimer may be either a homodimer (in case using two moleculesof a same TWSG-Fc fusion protein or polypeptide) or a heterodimer (incase using different fusion protein or polypeptides comprising differentsequences in their TWSG portions and/or Fc portions). In otherembodiments, multiple TWSG-Fc fusion proteins or polypeptides (ashomo-monomers or hetero-monomers) form a multimeric polypeptide complex.

In certain aspects, the present disclosure provides a composition orformulation comprising the TWSG-Fc fusion protein or polypeptidedescribed herein and a pharmaceutically acceptable carrier. In someembodiments, such composition or formulation is a pharmaceuticalcomposition or formulation.

In certain aspects, the present disclosure provides a composition orformulation comprising the multimeric polypeptide complex comprising theTWSG-Fc fusion protein or polypeptide described herein and apharmaceutically acceptable carrier. In some embodiments, suchcomposition or formulation is a pharmaceutical composition orformulation.

In other aspects, the present disclosure provides novel Twistedgastrulation (TWSG) polynucleotides, such as an isolated TWSGpolynucleotide or an at least partly purified TWSG polynucleotide. Insome embodiments, the present disclosure provides an isolated and atleast partly purified TWSG polynucleotide. For example, the presentdisclosure provides a polynucleotide encoding a TWSG-Fc fusion proteinor polypeptide as described herein. Such polynucleotide may comprise aDNA, an RNA, or an mRNA molecule.

In certain embodiments, the TWSG portion of the polynucleotide comprisesa nucleic acid sequence at least about 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the sequence of SEQID NO: 2. In some embodiments, the TWSG portion of the polynucleotidecomprises a nucleic acid sequence at least 80% identical to the sequenceof SEQ ID NO: 2. In some embodiments, the TWSG portion of thepolynucleotide comprises a nucleic acid sequence at least 85% identicalto the sequence of SEQ ID NO: 2. In some embodiments, the TWSG portionof the polynucleotide comprises a nucleic acid sequence at least 90%identical to the sequence of SEQ ID NO: 2. In some embodiments, the TWSGportion of the polynucleotide comprises a nucleic acid sequence at least95% identical to the sequence of SEQ ID NO: 2. In some embodiments, theTWSG portion of the polynucleotide comprises a nucleic acid sequence atleast 99% identical to the sequence of SEQ ID NO: 2. In someembodiments, the TWSG portion of the polynucleotide comprises a nucleicacid sequence of SEQ ID NO: 2. In some embodiments, the TWSG portion ofthe polynucleotide hybridizes, e.g., under highly stringent conditionsto a nucleic acid sequence that is complementary to the sequence of SEQID NO: 2. Such highly stringent conditions may comprise hybridization in6× sodium chloride/sodium citrate (SSC) at about 45° C. to about 65° C.,followed by at least one wash in about 0.2×SSC to about 2.0×SSC at about50° C. to about 65° C. In some embodiments, such highly stringentconditions may comprise hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 65° C., followed by a wash in 0.2×SSC at about65° C.

In some embodiments, the TWSG portion of the polynucleotide encodes aTWSG polypeptide comprising an amino acid sequence at least about 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to the sequence of SEQ ID NO: 8. In some embodiments, theencoded TWSG polypeptide comprises an amino acid sequence at least 80%identical to the sequence of SEQ ID NO: 8. In some embodiments, theencoded TWSG polypeptide comprises an amino acid sequence at least 85%identical to the sequence of SEQ ID NO: 8. In some embodiments, theencoded TWSG polypeptide comprises an amino acid sequence at least 90%identical to the sequence of SEQ ID NO: 8. In some embodiments, theencoded TWSG polypeptide comprises an amino acid sequence at least 95%identical to the sequence of SEQ ID NO: 8. In some embodiments, theencoded TWSG polypeptide comprises an amino acid sequence at least 99%identical to the sequence of SEQ ID NO: 8. In some embodiments, theencoded TWSG polypeptide comprises an amino acid sequence of SEQ ID NO:8.

In some embodiments, the TWSG portion of the polynucleotide comprises awild-type or naturally occurring nucleic acid sequence. In otherembodiments, the TWSG portion comprises at least one nucleic acidsubstitution relative to a wild-type or naturally occurring nucleic acidsequence. Such nucleic acid substitution may encode, for example, atleast one amino acid substitution in the TWSG polypeptide, such as apotential glycosylation site in order to, for example, remove apotential glycosylation site. Alternatively, such nucleic acidsubstitution may encode at least one amino acid substitution in theencoded TWSG polypeptide at a site potentially affecting the biologicalfunction and/or interaction affinity of the encoded TWSG polypeptide.

In some embodiments, the TWSG polypeptide encoded by the polynucleotidedescribed herein comprises a signal sequence, e.g., at the N-terminus ofthe encoded TWSG polypeptide. In other embodiments, the encoded TWSGpolypeptide does not comprise a signal sequence.

In some embodiments, the Fc portion of the polynucleotide describedherein hybridizes, e.g., under highly stringent conditions, to a nucleicacid sequence that is complementary to the sequence of a polynucleotideencoding a polypeptide comprising an amino acid sequence of SEQ ID NO:3, 4, 5, 6, or 7. Such highly stringent conditions may comprisehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C.to about 65° C., followed by at least one wash in about 0.2×SSC to about2.0×SSC at about 50° C. to about 65° C. In some embodiments, such highlystringent conditions may comprise hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 65° C., followed by a wash in0.2×SSC at about 65° C.

In some embodiments, the polynucleotide provided in the presentdisclosure hybridizes, e.g., under highly stringent conditions, to anucleic acid sequence that is complementary to the sequence of SEQ IDNO: 14. Such highly stringent conditions may comprise hybridization in6× sodium chloride/sodium citrate (SSC) at about 45° C. to about 65° C.,followed by at least one wash in about 0.2×SSC to about 2.0×SSC at about50° C. to about 65° C. In some embodiments, such highly stringentconditions may comprise hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 65° C., followed by a wash in 0.2×SSC at about65° C.

In some embodiments, the polynucleotide provided in the presentdisclosure hybridizes, e.g., under highly stringent conditions to anucleic acid sequence that is complementary to the sequence of apolynucleotide encoding a polypeptide comprising an amino acid sequenceof SEQ ID NO: 9. In other embodiments, the polynucleotide provided inthe present disclosure hybridizes, e.g., under highly stringentconditions to a nucleic acid sequence that is complementary to thesequence of a polynucleotide encoding a polypeptide comprising an aminoacid sequence of SEQ ID NO: 13. Such highly stringent conditions maycomprise hybridization in 6× sodium chloride/sodium citrate (SSC) atabout 45° C. to about 65° C., followed by at least one wash in about0.2×SSC to about 2.0×SSC at about 50° C. to about 65° C. In someembodiments, such highly stringent conditions may comprise hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 65° C., followed bya wash in 0.2×SSC at about 65° C.

In some embodiments, the polynucleotide of the present disclosurecomprises wild-type or naturally occurring nucleic acid sequences for atleast one of the TWSG portion and the Fc portion. In some embodiments,the polynucleotide comprises at least one nucleic acid substitutionrelative to wild-type or naturally occurring nucleic acid sequences.Such nucleic acid substitution may encode, for example, at least oneamino acid substitution in the encoded TWSG-Fc fusion polypeptide. Forexample, the nucleic acid substitution may encode at least one aminoacid substation in at least one potential glycosylation site in orderto, for example, remove a potential glycosylation a potentialglycosylation site. Alternatively, such nucleic acid substitution mayencode at least one amino acid substitution in the encodable TWSG or Fcportion at at least one site potentially affecting the biologicalfunction and/or interaction affinity of such portion. In someembodiments, such polynucleotide comprises at least one nucleic acidsubstitution to SEQ ID NO: 14.

In certain aspects, the present disclosure provides a composition orformulation comprising a polynucleotide as described herein and apharmaceutically acceptable carrier. In some embodiments, suchcomposition or formulation is a pharmaceutical composition orformulation.

In certain aspects, the present disclosure provides a vector comprisingat least one regulatory sequence operably linked to the polynucleotideencoding a TWSG-Fc fusion protein or polypeptide as described herein. Insome embodiments, the vector is an expression vector (e.g., a plasmid)or a viral vector (e.g., an adenoviral (AV) vector or an advancedadenoviral (AAV) vector). In some embodiments, the expressed TWSG-Fcfusion protein or polypeptide forms a multimeric polypeptide complexdescribed herein.

In certain aspects, the present disclosure provides a host cellcomprising, preferably expressing a vector described herein. In someembodiments, the polynucleotide encoding a TWSG-Fc fusion protein orpolypeptide described herein is produced in the host cell by expressingthe vector. In some embodiments, the TWSG-Fc fusion protein orpolypeptide produced in such host cell forms a multimeric polypeptidecomplex described herein. Such host cell may be any suitable cell, suchas a eukaryotic cell or a bacterial cell. In some embodiments, the hostcell is a mammalian cell, a vertebrate cell, a yeast cell, or an insectcell. In some embodiments, the host cell is a Chinese hamster ovary(CHO) cell.

In certain aspects, the present disclosure provides a kit or articlecomprising at least one of the TWSG-Fc fusion protein or polypeptide,the polynucleotide encoding such TWSG-Fc fusion protein or polypeptide,the composition, the vector, and the host cell described herein.Optionally, the kit or article may also comprise an administrationdevice (e.g., an infusion device, an injection device, an inhale device,a nebulizer device, an implantable device, etc.) to administer suchfusion protein or polypeptide, polynucleotide, composition, vector,and/or host cell to a subject (e.g., a mammal, such as a human).Optionally, the kit or article may also contain instructions for suchadministration. In some embodiments, the administered TWSG-Fc fusionprotein or polypeptide produced forms a multimeric polypeptide complexdescribed herein.

In certain aspects, the present disclosure provides a non-human animalengineered to express, or to overexpress, the TWSG-Fc fusion protein orpolypeptide, and/or the polynucleotide encoding the TWSG-Fc fusionprotein or polypeptide, as described herein. In some embodiments, suchnon-human animal is genetically engineered. In some embodiments, theexpressed or overexpressed TWSG-Fc fusion protein or polypeptide forms amultimeric polypeptide complex described herein in such non-humananimal.

In certain aspects, the present disclosure provides a method ofproducing a TWSG-Fc fusion protein or polypeptide, comprising:

i) providing a cell comprising a polynucleotide encoding the TWSG-Fcfusion protein or polypeptide, as described herein; and

ii) culturing the cell under conditions suitable for expression of theTWSG-Fc fusion polypeptide encoded by the polynucleotide, and optionally

-   -   iii) recovering the expressed TWSG-Fc fusion polypeptide.

In some embodiments, the TWSG-Fc fusion protein or polypeptide producedthrough such method forms a multimeric polypeptide complex describedherein.

Any suitable methods of protein production in cell culture may be usedherein. In some embodiments, the cell for producing TWSG-Fc is amammalian cell, such as a CHO cell.

In certain aspects, the present disclosure provides a method ofinhibiting BMP signaling in a cell, tissue, or organ, comprisingcontacting the cell, tissue, or organ with a TWSG-Fc fusion protein orpolypeptide, multimeric polypeptide complex, polynucleotide,composition, vector, and/or host cell, as described herein.

In certain aspects, the present disclosure provides a method ofincreasing red blood cell and/or hemoglobin levels, or reducing bloodtransfusion-dependence (TD), in a subject, comprising administering tothe subject a TWSG-Fc fusion protein or polypeptide, multimericpolypeptide complex, polynucleotide, composition, vector, and/or hostcell, as described herein.

In certain aspects, the present disclosure provides a method ofincreasing iron levels in a subject, comprising administering to thesubject a TWSG-Fc fusion protein or polypeptide, multimeric polypeptidecomplex, polynucleotide, composition, vector, and/or host cell, asdescribed herein. In some embodiments, the increase of iron level in thesubject occurs in the spleen. In some embodiments, the increase of ironlevel in the subject occurs in spleen but no other tissues.

In certain aspects, the present disclosure provides a method of treatingiron overload in a subject, comprising administering to the subject aTWSG-Fc fusion protein or polypeptide, multimeric polypeptide complex,polynucleotide, composition, vector, and/or host cell, as describedherein.

In certain aspects, the present disclosure provides a method ofpreventing iron overload in a subject, comprising administering to thesubject a TWSG-Fc fusion protein or polypeptide, multimeric polypeptidecomplex, polynucleotide, composition, vector, and/or host cell, asdescribed herein.

In certain aspects, the present disclosure provides a method of treatingdysregulation of BMP signaling in a subject, comprising administering tothe subject a TWSG-Fc fusion protein or polypeptide, multimericpolypeptide complex, polynucleotide, composition, vector, and/or hostcell, as described herein. In some embodiments, the dysregulation of BMPsignaling results in at least one of anemia, splenomegaly,erythroblast-induced bone pathology, iron overload, and thalassemiasyndromes in the subject. In some embodiments, the dysregulation of BMPsignaling results in ineffective erythropoiesis in the subject. In someembodiments, treating the dysregulation of BMP signaling results intreating ineffective erythropoiesis in the subject. In some embodiments,treating the dysregulation of BMP signaling results in treating at leastone of anemia, splenomegaly, erythroblast-induced bone pathology, ironoverload, and thalassemia syndromes.

In certain aspects, the present disclosure provides a method of treatinganemia in a subject, comprising administering to the subject a TWSG-Fcfusion protein or polypeptide, multimeric polypeptide complex,polynucleotide, composition, vector, and/or host cell, as describedherein.

In certain aspects, the present disclosure provides a method of treatinganemia in a subject, comprising administering to the subject a fusionprotein comprising a Twisted Gastrulation (TWSG) polypeptide and afragment crystallizable region (Fc) polypeptide, or a multimericpolypeptide complex comprising such fusion protein. In some embodiments,such fusion protein comprises the sequence of SEQ ID NO: 9. In otherembodiments, such fusion protein comprises the sequence of SEQ ID NO:13.

In certain aspects, the present disclosure provides a method of treatinganemia in a subject, comprising administering to the subject apolynucleotide encoding a fusion protein comprising a TwistedGastrulation (TWSG) polypeptide and a fragment crystallizable region(Fc) polypeptide. For example, such polynucleotide may comprise thesequence of SEQ ID NO: 14. In some embodiments, such fusion proteincomprises the sequence of SEQ ID NO: 9. In other embodiments, suchfusion protein comprises the sequence of SEQ ID NO: 13. In anon-limiting example, such fusion protein forms a multimeric polypeptidecomplex described herein in the subject.

In some embodiments, the anemia described herein comprises inheritedanemia, acquired anemia, anemia of chronic disease or inflammation,dyserythropoietic anemia (Types I and II), sickle cell anemia,hereditary spherocytosis, pyruvate kinase deficiency-related anemia, ormegaloblastic anemia. The anemia may be any anemia-related disease ordisorder described in the present disclosure.

Any suitable method may be used for administration in the therapeuticmethods described herein. For example, the administration may be throughat least one of topical, enteral, and parenteral routes. In someembodiments, the administration is through at least one of oral andnasal routes. In other embodiments, the administration is through atleast one of intravenous, subcutaneous, intraarterial, intraperitoneal,and intramuscular routes.

In certain aspects, the present disclosure provides a therapeuticmethod, as described herein, further comprising conjointly administeringa second pharmaceutical agent or second therapeutic practice. Suchsecond pharmaceutical agent may be, for example, EPO or an agonist oranalog thereof, or another BMP antagonist. Such second therapeuticpractice may be, for example, blood transfusion.

In certain aspects, the present disclosure provides a TWSG-Fc fusionprotein or polypeptide, a multimeric polypeptide complex comprising suchfusion protein or polypeptide, a polynucleotide encoding such fusionprotein or polypeptide, a composition comprising such fusion protein orpolypeptide or such polynucleotide, a vector comprising suchpolynucleotide, and/or a host cell expressing such vector, as describedherein, for inhibiting BMP signaling, increasing red blood cell and/orhemoglobin levels, reducing blood transfusion-dependence (TD),increasing iron levels, treating or preventing iron overload, ortreating dysregulation of BMP signaling in a subject.

In one aspect, the present disclosure provides a TWSG-Fc fusion proteinor polypeptide, a multimeric polypeptide complex comprising such fusionprotein or polypeptide, a polynucleotide encoding such fusion protein orpolypeptide, a composition comprising such fusion protein or polypeptideor such polynucleotide, a vector comprising such polynucleotide, and/ora host cell expressing such vector, as described herein, for treatinganemia in a subject.

In certain aspects, the present disclosure provides a use of a TWSG-Fcfusion protein or polypeptide, a multimeric polypeptide complexcomprising such fusion protein or polypeptide, a polynucleotide encodingsuch fusion protein or polypeptide, a composition comprising such fusionprotein or polypeptide or such polynucleotide, a vector comprising suchpolynucleotide, and/or a host cell expressing such vector, as describedherein, for inhibiting BMP signaling, increasing red blood cell and/orhemoglobin levels, reducing blood transfusion-dependence (TD),increasing iron levels, treating or preventing iron overload, ortreating dysregulation of BMP signaling in a subject.

In one aspect, the present disclosure provides a use of a TWSG-Fc fusionprotein or polypeptide, a multimeric polypeptide complex comprising suchfusion protein or polypeptide, a polynucleotide encoding such fusionprotein or polypeptide, a composition comprising such fusion protein orpolypeptide or such polynucleotide, a vector comprising suchpolynucleotide, and/or a host cell expressing such vector, as describedherein, for treating anemia in a subject.

In certain aspects, the present disclosure provides a use of a TWSG-Fcfusion protein or polypeptide, a multimeric polypeptide complexcomprising such fusion protein or polypeptide, a polynucleotide encodingsuch fusion protein or polypeptide, a composition comprising such fusionprotein or polypeptide or such polynucleotide, a vector comprising suchpolynucleotide, and/or a host cell expressing such vector, as describedherein, the manufacture of a medicament for the treatment of inhibitingBMP signaling, increasing red blood cell and/or hemoglobin levels,reducing blood transfusion-dependence (TD), increasing iron levels,treating or preventing iron overload, or treating dysregulation of BMPsignaling in a subject.

In one aspect, the present disclosure provides a use of a TWSG-Fc fusionprotein or polypeptide, a multimeric polypeptide complex comprising suchfusion protein or polypeptide, a polynucleotide encoding such fusionprotein or polypeptide, a composition comprising such fusion protein orpolypeptide or such polynucleotide, a vector comprising suchpolynucleotide, and/or a host cell expressing such vector, as describedherein, for the manufacture of a medicament for the treatment of anemiain a subject.

The subject of the various methods and uses described herein ispreferably a mammal, most preferably a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of processed human TWSG (SEQ IDNO: 8). Dotted underline denotes potential N-glycosylation sites.

FIG. 2 depicts the amino acid sequence of processed human TWSG-Fc (SEQID NO: 9). Solid underline indicates the linker between the TWSG portionand the Fc portion.

FIG. 3 depicts the amino acid sequence of unprocessed human TWSG-Fc (SEQID NO: 13). Solid underlines indicate the signal sequence in theN-terminus of TWSG-Fc and the linker between the TWSG portion and the Fcportion.

FIG. 4 depicts a nucleotide sequence encoding unprocessed human TWSG-Fc(SEQ ID NO: 14).

FIG. 5 shows the effect of human TWSG-Fc vs. vehicle control oncirculating iron concentrations in mice. Compared to vehicle, treatmentof wild-type mice with TWSG-Fc for 1 week increased serum iron by 12%.Data are means±SEM; n=5 per group; **P<0.01.

FIG. 6 shows the effect of human TWSG-Fc (labeled as “TWSG-G1Fc”; rightpanel) vs. vehicle control (left panel) on iron accumulation in mousespleen as determined by microscopy with Perls' Prussian blue staining.Spleen sections from wild-type mice treated with TWSG-Fc for 4 weekscontained numerous deposits of iron reaction product spread acrossregions of active erythropoiesis known as red pulp (right panel). Littleor no staining was present in spleen sections from vehicle-treated mice(left panel). Magnification, 200×.

FIG. 7 shows effects of human TWSG-Fc (labeled as “TWSG-G1Fc”) vs.vehicle control on RBC count (FIG. 7A) and hemoglobin concentration(FIG. 7B) in mice. Compared to vehicle, treatment of wild-type mice withTWSG-Fc for 4 weeks increased RBC count by 15% (A) and hemoglobinconcentration by 12% (B). Data are means±SEM; n=5 per group; **P<0.01,***P<0.001.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

In part, the present disclosure provides novel TWSG polypeptides topromote iron availability and increased levels of red blood cells and/orhemoglobin in animals. TWSG is a secreted protein that regulatessignaling by certain bone morphogenetic proteins (BMPs), which are aprominent group of ligands in the superfamily that also includestransforming growth factor-β (TGF-β), growth differentiation factors(GDFs), and activins/inhibins. This superfamily contains a variety ofgrowth factors that share common sequence elements and structuralmotifs. These proteins are known to exert biological effects on a largevariety of cell types during embryogenesis as well as postnatally inboth vertebrates and invertebrates. Members of the superfamily performimportant functions during embryonic development in pattern formationand tissue specification and can influence a variety of differentiationprocesses, including adipogenesis, myogenesis, chondrogenesis,cardiogenesis, hematopoiesis, neurogenesis, and epithelial celldifferentiation. By manipulating the activity of a member of the TGF-βfamily, it is often possible to cause significant physiological changesin an organism. For example, the Piedmontese and Belgian Blue cattlebreeds carry a loss-of-function mutation in the gene encoding myostatin(MSTN; also called GDF8) that causes a marked increase in muscle mass.Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans,inactive alleles of MSTN are associated with increased muscle mass and,reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004,350:2682-8.

TGF-β superfamily signaling is mediated by heteromeric complexes of typeI and type II serine/threonine kinase receptors, which phosphorylate andactivate downstream Smad proteins upon ligand stimulation (Massague,2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type IIreceptors are transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine specificity.Ligand binding triggers formation of a heteromeric complex of type I andtype II receptors, phosphorylation of type I receptor by type IIreceptor, and subsequent activation of Smad1/5/8 or Smad2/3transcription factors, which then act in the nucleus to regulate geneexpression.

Twisted gastrulation (abbreviated TWSG or TWSG1 in mammals and Tsg innonmammals; collectively as “TWSG” in the instant disclosure) is ahighly conserved, secreted protein implicated as an important regulatorof BMP signaling during embryogenesis, particularly based on studies inthe fruit fly (Drosophila) and frog (Xenopus) (Mason et al., 1994, GenesDev 8:1489-1501; Graf et al., 2001, Mamm Genome 12:554-560; Zakin etal., 2010, Curr Biol 20:R89-92). TWSG is often classified in a diversegroup of extracellular proteins having the shared ability to antagonizeBMP signaling (Avsian-Kretchmer et al., 2004, Mol Endocrinol 18:1-12;Gazzerro et al., 2006, Rev Endocr Metab Disord 7:51-65; Walsh et al.,2010, Trends Cell Biol 20:244-256). However, embryogenic studiesindicate that TWSG/Tsg can either promote (Oelgeschläger et al., 2000,Nature 405:757-763; Little et al., 2004, Development 131:5825-5835; Xieet al., 2004, Development 132:383-391) or inhibit (Scott et al., 2001,Nature 410:475-478; Ross et al., 2001, Nature 410:479-483; Chang et al.,2001, Nature 410:483-487) BMP signaling in a context-dependent mannerinvolving direct BMP binding as well as interaction with other proteinssuch as chordin (Larrain et al., 2001, Development 128:4439-4447). Whenchordin levels are low, the binary complex of TWSG/Tsg with BMP isthought to be permissive for BMP signaling by maintaining ligandsolubility (De Robertis et al., 2000, Nat Rev Genet 1:171-181; Zakin etal., 2010, Curr Biol 20:R89-92; Rider et al., 2010, Biochem J 429:1-12).To date, the specific ligands shown to bind TWSG/Tsg are BMP2, BMP4, andBMP7 (Oelgeschläger et al., 2000, Nature 405:757-763; Chang et al.,2001, Nature 410:483-487; Zakin et al., 2005, Development132:2489-2499).

Like other ligands in the TGFβ superfamily, BMPs contain acharacteristic cysteine knot motif and are secreted as precursormolecules containing a larger N-terminal prodomain that is removed byproteolytic cleavage to generate mature dimeric ligand (Harrison et al.,Growth Factors 29:174, 2011; Shi et al., Nature 474:343, 2011). Althoughinitially named for their generally shared ability to induce boneformation (Cheng et al., 2003, J Bone Joint Surg Am 85-A:1544-1552),BMPs are now recognized to play critical roles in early embryogenesisand to exhibit a broad spectrum of biological activities in later stagesof development (Hogan et al., 1996, Genes Dev 10:1580-1594; Gazzerro etal., 2006, Rev Endocr Metab Disord 7:51-65).

Gene knockout studies in mice have been important in identifyingdevelopmental and/or physiologic roles of individual BMPs in mammals forwhich other BMPs cannot effectively substitute (Chang et al., 2002,Endocr Rev 23:787-823). BMP2 and BMP4 are closely related, and globalloss of function of either gene causes early embryonic lethality in mice(Winnier et al., 1995, Genes Dev 9:2105-2116; Zhang et al., 1996,Development 122:2977-2986). Studies with BMP2/BMP4 compound mutant micehave revealed functions of BMP2 in development of multiple organsystems, including the skeleton, heart, eye, ventral body wall, andplacenta (Goldman et al., 2009, Mech Develop 126:117-127). Whereas BMP2,BMP4, BMP7, and the closely related BMP6 can all stimulate boneformation (Cheng et al., 2003, J Bone Joint Surg Am 85-A:1544-1552),conditional ablation of these individual genes produces markedlydifferent outcomes. Combined conditional knockout of BMP2 and BMP4 inembryonic limb buds causes severe impairment of osteogenesis(Bandyopadhyay et al., 2006, PLOS Genet 2:e216). Conditional knockout ofBMP2 in limb bones leads to spontaneous fractures in mice and reveals anobligatory role for BMP2 in fracture healing (Tsuji et al., 2006, NatGenet 38:1424-1429), whereas conditional knockout of BMP7 has no effecton postnatal limb growth or maintenance of bone mass, thus indicatingthat other factors present in adult bone can compensate for its absence(Tsuji et al., 2010, J Orthop Res 28:384-389). Endogenous TWSG has beenimplicated as a negative regulator of pro-osteoclastic BMP2 signaling inmice (Pham et al., 2011, J Cell Biochem 112:793-803). BMP2, but notBMP4, also serves a critical role in cartilage formation during bonedevelopment (Shu et al., 2011, J Cell Sci 124:3428-3440). Finally, BMP6ablation in mice does not cause major skeletal defects (Solloway et al.,1998, Dev Genet 22:321-339) but does alter bone morphometry consistentwith a non-redundant role for BMP6 in periosteal bone formation (Perryet al., 2008, Bone 42:216-225).

Significantly, BMP4 and BMP6 are implicated in postnatal erythropoiesisand/or iron homeostasis. The body has evolved sophisticated mechanismsfor regulating iron levels because dietary iron is undependable and lowplasma iron levels limit iron uptake and hemoglobin synthesis byerythroid precursors, causing anemia. On the other hand, excessiveaccumulation of iron (iron overload) causes damage to cells and tissuesdue in part to generation of reactive oxygen species (Fibach et al.,2008, Curr Mol Med 8:609-619). The hepatic peptide hormone hepcidin isnow widely considered to be the master regulator of iron homeostasis dueto its ability to promote degradation of the iron-transport proteinferroportin which is otherwise present on the surface of iron-absorbingenterocytes, iron-recycling macrophages, and iron-storing hepatocytes(Ganz et al., 2012, Biochim Biophys Acta 1823:1434-1443). In mice, BMP6and its co-receptor hemojuvelin [encoded by the hemochromatosis type 2gene (HFE2) and also known as repulsive guidance molecule C (RGMc)] areessential for normal iron homeostasis since their loss interferes withthe hepcidin response to iron loading (Niederkofler et al., 2005, J ClinInvest 115:2180-2186; Babitt et al., 2006, Nat Genet 38:531-539;Camaschella, 2009, Nat Genet 41:386-388; Meynard et al., 2009, Nat Genet41:478-481; Andriopoulos et al., 2009, Nat Genet 41:482-487). OtherBMPs, including BMP2, BMP4, BMP7, and BMP9, are able to stimulatehepcidin expression in vitro (Xia et al., 2008, Blood 111:5195-5204),but their physiologic relevance has not been established (Ganz et al.,2012, Biochim Biophys Acta 1823:1434-1443).

In addition to BMP6 involvement in iron homeostasis, BMP4 has beenimplicated as an important ligand in a stress-erythropoiesis pathway inmice, which mediates induction of compensatory erythropoiesis inresponse to acute and chronic anemia (Paulson et al., 2011, Curr OpinHematol 18:139-145). This stress-erythropoiesis pathway is distinct fromsteady-state erythropoiesis and occurs in mice in the fetal liver, adultliver, and adult spleen. In this pathway, BMP4 is thought to promote theexpansion of a population of specialized stress-erythroid progenitors inresponse to an acute anemia stimulus, and therefore inhibition of BMP4signaling by itself would be predicted to block thestress-erythropoiesis response and be counterproductive in treatinganemia.

Specific BMPs shown to bind to TWSG are restricted in which combinationsof receptor heterodimers they use. BMP2 and BMP4 signal through the typeI receptors activin receptor-like kinase-3 (ALK3 or BMPRIA) and ALK6(BMPRIB), and through the type II receptors BMP receptor type II(BMPRII), activin receptor type IIA (ActRIIA or ACVR2A), and activinreceptor type IIB (ActRIIB or ACVR2B) (Mueller et al., 2012, FEBS Lett586:1846-1859). In addition to heteromeric complexes containing thesereceptors, BMP6 and BMP7 can signal through combinations containing ALK2(ActRIA or ACVR1) as well. Upon ligand-induced formation of heteromericreceptor complex, ALK2, ALK3, and ALK6 typically activate the Smad1/5/8subfamily of intracellular effectors to regulate gene expression(Miyazono et al., 2010, J Biochem 147:35-51).

As demonstrated herein, a TWSG polypeptide selectively inhibitssignaling by certain BMPs, unexpectedly increases red blood cell levelsin mice in vivo, and exerts beneficial effects on systemic ironavailability in vivo that support its promotion of red blood cellformation. Effects of TWSG polypeptides may be particularly useful intreating anemia of inflammation (also known as anemia of chronicdisease), which results when inflammatory processes increase hepcidinexpression and thereby limit iron availability for erythropoiesis. Itshould be noted that erythropoiesis is a complex process, regulated by avariety of factors, including EPO, G-CSF, and iron homeostasis. Theterms “increase red blood cell levels” and “promote red blood cellformation” refer to clinically observable metrics, such as hematocrit,red blood cell counts and hemoglobin measurements, and are intended tobe neutral as to the mechanism by which such changes occur.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. The term “sequencesimilarity,” in all its grammatical forms, refers to the degree ofidentity or correspondence between nucleic acid or amino acid sequencesthat may or may not share a common evolutionary origin. However, incommon usage and in the instant application, the term “homologous,” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and may or may not relate to a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ±10%. Alternatively, andparticularly in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably <5-fold and more preferably <2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

2. TWSG Polypeptides

In certain aspects, the invention relates to TWSG polypeptides, e.g.,soluble TWSG polypeptides, comprising, for example, fragments,functional variants, and modified forms of wild-type TWSG polypeptides.In certain embodiments, the TWSG polypeptides have the same or at leastone similar biological activity as a corresponding wild-type TWSGpolypeptide. For example, a TWSG polypeptide of the invention may bindto and inhibit the function of a BMP ligand (e.g., BMP2, BMP4, BMP6,BMP7, or BMP9). Optionally, a TWSG polypeptide increases ironavailability, red blood numbers, and/or circulating hemoglobinconcentrations. Examples of TWSG polypeptides include human TWSGprecursor polypeptides (SEQ ID NO: 1) having one or more sequencevariations, and soluble human TWSG polypeptides (e.g., SEQ ID NOs: 8, 9,and 13) having one or more sequence variations.

As used herein, the term “TWSG” refers to twisted gastrulation proteinsfrom any species and variants derived from such TWSG proteins bymutagenesis or other modification. Reference to TWSG herein isunderstood to be a reference to any one of the currently identifiedforms. Members of the TWSG family are secreted proteins composed of anN-terminal domain that is essential for BMP binding and a C-terminaldomain that interacts with chordin and other proteins. The amino acidsequence of mature native human TWSG (SEQ ID NO: 8) is illustrated inFIG. 1.

The term “TWSG polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a TWSG family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. For example, TWSGpolypeptides include polypeptides derived from the sequence of any knownTWSG having a sequence at least about 80% identical to the sequence ofan TWSG polypeptide, and optionally at least 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity. For example, a TWSGpolypeptide may bind to and inhibit the function of a BMP and/or otherligand in the TGFβ superfamily. A TWSG polypeptide may be selected foractivity in promoting red blood cell formation in vivo. Examples of TWSGpolypeptides include human TWSG precursor polypeptide (SEQ ID NO: 1) andsoluble human TWSG polypeptides (e.g., SEQ ID NOs: 8, 9, and 13).Numbering of amino acids for all TWSG polypeptides described herein isbased on the numbering for SEQ ID NO: 1, unless specifically designatedotherwise.

The human TWSG precursor sequence (NCBI Reference Sequence NP 065699.1)is as follows:

(SEQ ID NO: 1) 1 MKLHYVAVLT LAILMFLTWL PESLSCNKAL CASDVSKCLI QELCQCRPGE51 GNCSCCKECM LCLGALWDEC CDCVGMCNPR NYSDTPPTSK STVEELHEPI 101PSLFRALTEG DTQLNWNIVS FPVAEELSHH ENLVSFLETV NQPHHQNVSV 151PSNNVHAPYS SDKEHMCTVV YEDDCMSIHQ CKISCESMGA SKYRWFHNAC 201CECIGPECID YGSKTVKCMN CMF

The leader (signal) sequence and three potential N-linked glycosylationsites are underlined.

A nucleotide sequence encoding human TWSG precursor (nucleotides 192-860of NCBI Reference Sequence NM_020648.5) is as follows:

(SEQ ID NO: 2) 1 ATGAAGTTAC ACTATGTTGC TGTGCTTACT CTAGCCATCC TGATGTTCCT51 GACATGGCTT CCAGAATCAC TGAGCTGTAA CAAAGCACTC TGTGCTAGTG 101ATGTGAGCAA ATGCCTCATT CAGGAGCTCT GCCAGTGCCG GCCGGGAGAA 151GGCAATTGCT CCTGCTGTAA GGAGTGCATG CTGTGTCTTG GGGCCCTTTG 201GGACGAGTGC TGTGACTGTG TTGGTATGTG TAATCCTCGA AATTATAGTG 251ACACACCTCC AACTTCAAAG AGCACAGTGG AGGAGCTGCA TGAACCGATC 301CCTTCTCTCT TCCGGGCACT CACAGAAGGA GATACTCAGT TGAATTGGAA 351CATCGTTTCT TTCCCTGTTG CAGAAGAACT TTCACATCAT GAGAATCTGG 401TTTCATTTTT AGAAACTGTG AACCAGCCAC ACCACCAGAA TGTGTCTGTC 451CCCAGCAATA ATGTTCACGC GCCTTATTCC AGTGACAAAG AACACATGTG 501TACTGTGGTT TATTTTGATG ACTGCATGTC CATACATCAG TGTAAAATAT 551CCTGTGAGTC CATGGGAGCA TCCAAATATC GCTGGTTTCA TAATGCCTGC 601TGCGAGTGCA TTGGTCCAGA ATGTATTGAC TATGGTAGTA AAACTGTCAA 651ATGTATGAAC TGCATGTTT

In some embodiments, TWSG polypeptides comprise a signal sequence inaddition to the TWSG protein. The signal sequence can be a native signalsequence of a TWSG protein, or a signal sequence from another protein,such as a tissue plasminogen activator (TPA) signal sequence or a honeybee melittin (HBM) signal sequence.

TWSG is highly conserved in amino acid sequence across vertebrates,including human, chimpanzee, rhesus macaque, dog, cow, mouse, rat,chicken, zebrafish, and frog. See, e.g., Oelgeschlager et al., 2000,Nature 405:757-763. Ligands that bind to TWSG are also highly conserved.Accordingly, comparisons of TWSG sequences from various vertebrateorganisms provide insights into residues that may be altered. Therefore,an active, variant human TWSG polypeptide may include one or more aminoacids at corresponding positions from the sequence of another vertebrateTWSG polypeptide, or may include a residue that is similar to that inthe human or other vertebrate sequence.

In certain embodiments, isolated fragments of TWSG polypeptides can beobtained by screening polypeptides recombinantly produced from thecorresponding fragment of the nucleic acid encoding an TWSG polypeptide(e.g., SEQ ID NOs: 2 and 14). In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. The fragments can beproduced (recombinantly or by chemical synthesis) and tested to identifythose peptidyl fragments that can function, for example, as antagonists(inhibitors) or agonists (activators) of a TWSG polypeptide or a TWSGpolypeptide ligand.

In certain embodiments, the TWSG polypeptide is a variant having anamino acid sequence that is at least 75% identical to an amino acidsequence selected from SEQ ID NOs: 1, 8, 9, or 13. In certain cases, theTWSG polypeptide has an amino acid sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 8, 9, or 13. In certainembodiments, the TWSG polypeptide comprises, consists essentially of, orconsists of, an amino acid sequence at least 80%, 85%, 90%, 95%, 97%,98%, 99% or 100% identical to an amino acid sequence selected from SEQID NOs: 1, 8, 9, or 13.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of a TWSG polypeptide forsuch purposes as enhancing therapeutic efficacy, or stability (e.g., exvivo shelf life and resistance to proteolytic degradation in vivo). TWSGpolypeptides can also be produced by amino acid substitution, deletion,or addition. For instance, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (e.g., conservativemutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Whether a change in the amino acid sequence of a TWSGpolypeptide results in a functional variant can be readily determined byassessing the ability of the TWSG polypeptide to produce a response incells relative to wild-type TWSG polypeptide, or to bind to one or moreligands, such as BMP2, BMP4, BMP6, BMP7, or BMP9 as compared towild-type TWSG polypeptide.

In certain specific embodiments, the present invention contemplatesmaking mutations in a TWSG polypeptide such that the TWSG polypeptidehas altered ligand-binding activities (e.g., binding affinity or bindingspecificity). In certain cases, such TWSG polypeptides have altered(elevated or reduced) binding affinity for a specific ligand. In othercases, the TWSG polypeptides have altered binding specificity for TWSGligands.

In certain embodiments, the present invention contemplates TWSGpolypeptides having specific mutations in TWSG so as to alter theglycosylation of the TWSG polypeptide. Exemplary glycosylation sites inTWSG polypeptides are illustrated in FIG. 1 (e.g., the underlinedNX(S/T) sites). Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (where“X” is any amino acid), which is specifically recognized by appropriatecellular glycosylation enzymes. The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the sequence of the wild-type TWSG polypeptide (for O-linkedglycosylation sites). A variety of amino acid substitutions or deletionsat one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a TWSG polypeptide is by chemical or enzymatic coupling ofglycosides to the TWSG polypeptide. Depending on the coupling mode used,the sugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 and in Aplin and Wriston (1981) CRCCrit. Rev. Biochem., pp. 259-306, incorporated by reference herein.Removal of one or more carbohydrate moieties present on a TWSGpolypeptide may be accomplished chemically and/or enzymatically.Chemical deglycosylation may involve, for example, exposure of the TWSGpolypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Chemical deglycosylation is further described by Hakimuddin et al.(1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal.Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on TWSGpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol.138:350. The sequence of a TWSG polypeptide may be adjusted, asappropriate, depending on the type of expression system used, asmammalian, yeast, insect and plant cells may all introduce differingglycosylation patterns that can be affected by the amino acid sequenceof the peptide. In general, TWSG polypeptides for use in humans will beexpressed in a mammalian cell line that provides proper glycosylation,such as HEK293 or CHO cell lines, although other mammalian expressioncell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of a TWSG polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying TWSG polypeptidesequences. The purpose of screening such combinatorial libraries may beto generate, for example, TWSG polypeptide variants which have alteredproperties, such as altered pharmacokinetics, or altered ligand binding.A variety of screening assays are provided below, and such assays may beused to evaluate variants. For example, a TWSG polypeptide variant maybe screened for the ability to bind to a TWSG polypeptide, to preventbinding of a TWSG ligand to a TWSG polypeptide, or to interfere withsignaling caused by a TWSG ligand.

The activity of a TWSG polypeptide or its variants may also be tested ina cell-based or in vivo assay. For example, the effect of a TWSGpolypeptide variant on the expression of genes involved in hematopoiesismay be assessed. This may, as needed, be performed in the presence ofone or more recombinant TWSG ligand proteins (e.g., BMP6), and cells maybe transfected so as to produce a TWSG polypeptide and/or variantsthereof, and optionally, a TWSG ligand. Likewise, a TWSG polypeptide maybe administered to a mouse or other animal, and one or more bloodmeasurements, such as an RBC count, hemoglobin levels, hematocrit levelsiron stores, or reticulocyte count may be assessed using art-recognizedmethods.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a reference TWSG polypeptide. Such variant proteins,when expressed from recombinant DNA constructs, can be used in genetherapy protocols. Likewise, mutagenesis can give rise to variants whichhave intracellular half-lives dramatically different than thecorresponding unmodified TWSG polypeptide. For example, the alteredprotein can be rendered either more stable or less stable to proteolyticdegradation or other processes which result in destruction of, orotherwise inactivation of an unmodified TWSG polypeptide. Such variants,and the genes which encode them, can be utilized to alter TWSGpolypeptide levels by modulating the half-life of the TWSG polypeptides.For instance, a short half-life can give rise to more transientbiological effects and, when part of an inducible expression system, canallow tighter control of recombinant TWSG polypeptide levels within thecell. In an Fc fusion protein, mutations may be made in the linker (ifany) and/or the Fc portion to alter the half-life of the protein.

In certain embodiments, the TWSG polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the TWSG polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. As a result,TWSG polypeptides may contain non-amino acid elements, such aspolyethylene glycols, lipids, poly- or mono-saccharide, and phosphates.Effects of such non-amino acid elements on the functionality of a TWSGpolypeptide may be tested as described herein for other TWSG polypeptidevariants. When a TWSG polypeptide is produced in cells by cleaving anascent form of the TWSG polypeptide, post-translational processing mayalso be important for correct folding and/or function of the protein.Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293)have specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the TWSG polypeptides.

In certain aspects, TWSG polypeptides include fusion proteins having atleast a portion of a TWSG polypeptide and one or more fusion domains.Well-known examples of such fusion domains include, but are not limitedto, polyhistidine, Glu-Glu, glutathione S transferase (GST),thioredoxin, protein A, protein G, an immunoglobulin heavy chainconstant region (e.g., an Fc), maltose binding protein (MBP), or humanserum albumin. A fusion domain may be selected so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆) (SEQ ID NO: 22) fusionpartners. As another example, a fusion domain may be selected so as tofacilitate detection of the TWSG polypeptides. Examples of suchdetection domains include the various fluorescent proteins (e.g., GFP)as well as “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation.

In certain preferred embodiments, a TWSG polypeptide is fused to adomain that stabilizes the TWSG polypeptide in vivo (a “stabilizer”domain). By “stabilizing” is meant anything that increases serumhalf-life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherincreasing red blood cell levels).

As specific examples, the present disclosure provides fusion proteinscomprising a TWSG polypeptide fused to a polypeptide comprising aconstant domain of an immunoglobulin, such as a CH1, CH2, or CH3 domainof an immunoglobulin or an Fc domain. Fc domains derived from humanIgG1, IgG2, IgG3, and IgG4 are provided herein. Other mutations areknown that decrease either CDC or ADCC activity, and collectively, anyof these variants are included in the disclosure and may be used asadvantageous components of a heteromultimeric complex of the disclosure.Optionally, the IgG1 Fc domain of SEQ ID NO: 3 has one or more mutationsat residues such as Asp-265, Lys-322, and Asn-434 (numbered inaccordance with the corresponding full-length IgG1). In certain cases,the mutant Fc domain having one or more of these mutations (e.g.,Asp-265 mutation) has reduced ability of binding to the Fcγ receptorrelative to a wildtype Fc domain. In other cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asn-434 mutation) hasincreased ability of binding to the MHC class I-related Fc-receptor(FcRN) relative to a wildtype Fc domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 3). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising amino acid sequences with 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3. Naturallyoccurring variants in G1Fc would include E134D and M136L according tothe numbering system used in SEQ ID NO: 3 (see Uniprot P01857).

(SEQ ID NO: 3) 1

51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 4). Dottedunderline indicates the hinge region and double underline indicatespositions where there are data base conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising amino acid sequences with 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% identity to SEQ ID NO: 4.

(SEQ ID NO: 4) 1

51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201CSVMHEALHN HYTQKSLSLS PGK

Two examples of amino acid sequences that may be used for the Fc portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 5) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 6) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptides comprisingamino acid sequences with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NOs: 5 and 6.

(SEQ ID NO: 5) 1

51 VSHEDPEVQF KWYVDGVEVH NAKTKPREEQ YNSTFRVVSV LTVLHQDWLN 101GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL 151TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS 201RWQQGNIFSC SVMHEALHNR FTQKSLSLSP GK (SEQ ID NO: 6) 1

51

101 EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLTV LHQDWLNGKE 151YKCKVSNKAL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL 201VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS KLTVDKSRWQ 251QGNIFSCSVM HEALHNRFTQ KSLSLSPGK

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del,F221Y when converted to the numbering system used in SEQ ID NO: 5, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01860].Specifically, variant WIS is lacking most of the V region and all of theCH1 region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CH1 region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 7). For example,see Uniprot P01861. Dotted underline indicates the hinge region. Inpart, the disclosure provides polypeptides comprising amino acidsequences with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 7.

(SEQ ID NO: 7) 1

51 EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE 101YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL 151VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ 201EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a TWSG polypeptide may be placed C-terminalto a heterologous domain, or, alternatively, a heterologous domain maybe placed C-terminal to a TWSG polypeptide. The TWSG polypeptide domainand the heterologous domain need not be adjacent in a fusion protein,and additional domains or amino acid sequences may be included C- orN-terminal to either domain or between the domains.

In certain embodiments, a TWSG fusion protein comprises an amino acidsequence as set forth in the formula A-B-C. The B portion is a TWSGpolypeptide comprising the amino acid sequence corresponding to aminoacids 26-223 of SEQ ID NO: 1. The A and C portions may be independentlyzero, one or more than one amino acids, and both the A and C portionswhen present are heterologous to B. The A and/or C portions may beattached to the B portion via a linker sequence. Exemplary linkersinclude short polypeptide linkers such as 2-10, 2-5, 2-4, 2-3 glycineresidues, such as, for example, a Gly-Gly-Gly linker. Other suitablelinkers are described herein above. In certain embodiments, a TWSGfusion protein comprises an amino acid sequence as set forth in theformula A-B-C, wherein A is a leader sequence, B consists of amino acids26-223 of SEQ ID NO: 1, and C is a polypeptide portion that enhances oneor more of in vivo stability, in vivo half life, uptake/administration,tissue localization or distribution, formation of protein complexes,and/or purification. In certain embodiments, a TWSG fusion proteincomprises an amino acid sequence as set forth in the formula A-B-C,wherein A is a TPA leader sequence, B consists of amino acids 26-223 ofSEQ ID NO: 1, and C is an immunoglobulin Fc domain. A preferred TWSGfusion protein comprises the amino acid sequence set forth in SEQ ID NO:13.

In certain embodiments, the TWSG polypeptides of the present inventioncontain one or more modifications that are capable of stabilizing theTWSG polypeptides. For example, such modifications enhance the in vitrohalf-life of the TWSG polypeptides, enhance circulatory half-life of theTWSG polypeptides or reduce proteolytic degradation of the TWSGpolypeptides. Such stabilizing modifications include, but are notlimited to, fusion proteins (including, for example, fusion proteinscomprising an TWSG polypeptide and a stabilizer domain), modificationsof a glycosylation site (including, for example, addition of aglycosylation site to a TWSG polypeptide), and modifications ofcarbohydrate moiety (including, for example, removal of carbohydratemoieties from a TWSG polypeptide). In the case of fusion proteins, aTWSG polypeptide is fused to a stabilizer domain such as an IgG molecule(e.g., an Fc domain). As used herein, the term “stabilizer domain” notonly refers to a fusion domain (e.g., Fc) as in the case of fusionproteins, but also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous polymer, such as polyethyleneglycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the TWSG polypeptides, which are isolated from,or otherwise substantially free of, other proteins.

In certain embodiments, TWSG polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such TWSG polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the TWSG polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary (CHO)cells, COS cells, baculovirus) as is well known in the art. In furtherembodiments, the modified or unmodified TWSG polypeptides may beproduced by digestion of recombinantly produced full-length TWSGpolypeptides by using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using commercially available software,e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be usedto identify proteolytic cleavage sites. Alternatively, such TWSGpolypeptides may be produced from recombinantly produced full-lengthTWSG polypeptides such as standard techniques known in the art, such asby chemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding TWSG Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the TWSG polypeptides disclosed herein.SEQ ID NO: 2 encodes a naturally occurring TWSG precursor polypeptidewhile SEQ ID NO: 14 encodes a soluble TWSG fusion protein. The subjectnucleic acids may be single-stranded or double stranded. Such nucleicacids may be DNA or RNA molecules. These nucleic acids may be used, forexample, in methods for making TWSG polypeptides or as directtherapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding TWSG polypeptidesare further understood to include nucleic acids that are variants of SEQID NOs: 2 and 14. Variant nucleotide sequences include sequences thatdiffer by one or more nucleotide substitutions, additions or deletions,such as allelic variants; and will, therefore, include coding sequencesthat differ from the nucleotide sequence of the coding sequencedesignated in SEQ ID NOs: 2 and 14.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 and 14.One of ordinary skill in the art will appreciate that nucleic acidsequences complementary to SEQ ID NO: 2 and 14, and variants of SEQ IDNO: 2 and 14, are also within the scope of this invention. In furtherembodiments, the nucleic acid sequences of the invention can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 2 and 14, complementsequence of SEQ ID NO: 2 and 14, or fragments thereof. As discussedabove, one of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In some embodiments, the invention provides nucleic acids whichhybridize under low stringency conditions of 6×SSC at room temperaturefollowed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 2 and 14 due to degeneracy in the genetic code are alsowithin the scope of the invention. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. In certain embodiments, the TWSGpolypeptide will be encoded by an alternative nucleotide sequence.Alternative nucleotide sequences are degenerate with respect to thenative TWSG nucleic acid sequence but still encode for the same fusionprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In certain preferred embodiments, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a TWSG polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the TWSG polypeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express. DNA sequences encoding a TWSG polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that: copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant TWSG polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In certain preferred embodiments, a vector will be designed forproduction of the subject TWSG polypeptides in CHO cells, such as aPcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors(Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison,Wis.). As will be apparent, the subject gene constructs can be used tocause expression of the subject TWSG polypeptides in cells propagated inculture, e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 2 or 14)for one or more of the subject TWSG polypeptides. The host cell may beany prokaryotic or eukaryotic cell. For example, a TWSG polypeptide ofthe invention may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present invention further pertains to methods ofproducing the subject TWSG polypeptides. For example, a host celltransfected with an expression vector encoding a TWSG polypeptide can becultured under appropriate conditions to allow expression of the TWSGpolypeptide to occur. The TWSG polypeptide may be secreted and isolatedfrom a mixture of cells and medium containing the TWSG polypeptide.Alternatively, the TWSG polypeptide may be retained cytoplasmically orin a membrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The subject TWSG polypeptides can be isolated from cell culture medium,host cells, or both, using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for particular epitopes of theTWSG polypeptides. In certain preferred embodiments, the TWSGpolypeptide is a fusion protein containing a domain which facilitatesits purification.

In other embodiments, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant TWSGpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified TWSG polypeptide (e.g., see Hochuliet al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In other embodiments, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Screening Assays

In certain aspects, the present invention relates to the use of thesubject TWSG polypeptides (e.g., soluble variant TWSG polypeptides) toidentify compounds (agents) which are agonist or antagonists of TWSGpolypeptides. Compounds identified through this screening can be testedto assess their ability to modulate red blood cell, hemoglobin and/orreticulocyte levels in vivo or in vitro. These compounds can be tested,for example, in animal models.

There are numerous approaches to screening for therapeutic agents forincreasing red blood cell or hemoglobin levels by targeting TWSGbioactivity. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturbTWSG-mediated effects on a selected cell line. In certain embodiments,the assay is carried out to screen and, identify compounds thatspecifically inhibit or reduce binding of a TWSG polypeptide to itsbinding partner, such as a TWSG ligand as disclosed herein (e.g., BMP2,BMP4, BMP6, BMP7, or BMP9). Alternatively, the assay can be used toidentify compounds that enhance binding of a TWSG polypeptide to itsbinding partner such as a TWSG ligand. In further embodiments, thecompounds can be identified by their ability to interact with a TWSGpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. Inspecific embodiments, the test agent is a small organic molecule havinga molecular weight of less than about 2,000 Daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatable crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a TWSGpolypeptide and its binding partner (e.g., a TWSG ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified TWSG polypeptide which is ordinarily capable of binding to aTWSG binding partner, as appropriate for the intention of the assay. Tothe mixture of the compound and TWSG polypeptide is then added to acomposition containing a TWSG binding partner. Detection andquantification of complexes between a TWSG polypeptide and its bindingpartner provides a means for determining the compound's efficacy atinhibiting (or potentiating) complex formation between the TWSGpolypeptide and its binding partner. The efficacy of the compound can beassessed by generating dose-response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. For example,in a control assay, isolated and purified TWSG ligand is added to acomposition containing the TWSG polypeptide, and the formation ofcomplexes between TWSG polypeptide and its binding partner arequantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the TWSG polypeptide and its binding partnermay be detected by a variety of techniques. For instance, modulation ofthe formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabeled (e.g., ³²P, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled TWSGpolypeptide or its binding partner, by immunoassay, or bychromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a TWSG polypeptide and its bindingpartner. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a TWSG polypeptide andits binding partner. See for example, U.S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two-hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between a TWSG polypeptide and its bindingpartner. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with a TWSG polypeptide. The interaction between thecompound and the TWSG polypeptide may be covalent or non-covalent. Forexample, such interaction can be identified at the protein level usingin vitro biochemical methods, including photo-crosslinking, radiolabeledligand binding, and affinity chromatography (Jakoby W B et al., 1974,Methods in Enzymology 46: 1). In certain cases, the compounds may bescreened in a mechanism based assay, such as an assay to detectcompounds which bind to a TWSG polypeptide. This may include a solidphase or fluid phase binding event. Alternatively, the gene encoding aTWSG polypeptide can be transfected with a reporter system (e.g.,β-galactosidase, luciferase, or green fluorescent protein) into a celland screened against the library preferably by a high throughputscreening or with individual members of the library. Other mechanismbased binding assays may be used, for example, binding assays whichdetect changes in free energy. Binding assays can be performed with thetarget fixed to a well, bead or chip or captured by an immobilizedantibody or resolved by capillary electrophoresis. The bound compoundsmay be detected usually using colorimetric or fluorescence or surfaceplasmon resonance.

5. Exemplary Therapeutic Uses

In certain embodiments, the TWSG polypeptides of the present inventioncan be used to increase red blood cell levels in mammals such as rodentsand primates, and particularly human patients. TWSG polypeptides,optionally combined with an EPO receptor activator, can be useful fortreating ineffective erythropoiesis. Originally distinguished fromaplastic anemia, hemorrhage, or peripheral hemolysis on the basis offerrokinetic studies (Ricketts et al., 1978, Clin Nucl Med 3:159-164),ineffective erythropoiesis describes a diverse group of anemias in whichproduction of mature RBCs is less than would be expected given thenumber of erythroid precursors (erythroblasts) present in the bonemarrow (Tanno et al., 2010, Adv Hematol 2010:358283). In such anemias,tissue hypoxia persists despite elevated erythropoietin levels due toineffective production of mature RBCs. A vicious cycle eventuallydevelops in which elevated erythropoietin levels drive massive expansionof erythroblasts, potentially leading to splenomegaly (spleenenlargement) due to extramedullary erythropoiesis (Aizawa et al, 2003,Am J Hematol 74:68-72), erythroblast-induced bone pathology (Di Matteoet al, 2008, J Biol Regul Homeost Agents 22:211-216), and tissue ironoverload, even in the absence of therapeutic RBC transfusions (Pippardet al, 1979, Lancet 2:819-821). Thus, by boosting erythropoieticeffectiveness, a TWSG polypeptide may break the aforementioned cycle andmay alleviate not only the underlying anemia but also the associatedcomplications of elevated erythropoietin levels, splenomegaly, bonepathology, and tissue iron overload. TWSG polypeptides can treatineffective erythropoiesis, including anemia and elevated EPO levels, aswell as complications such as splenomegaly, erythroblast-induced bonepathology, and iron overload, and their attendant pathologies. Withsplenomegaly, such pathologies include thoracic or abdominal pain andreticuloendothelial hyperplasia. Extramedullary hematopoiesis can occurnot only in the spleen but potentially in other tissues in the form ofextramedullary hematopoietic pseudotumors (Musallam et al., 2012, ColdSpring Harb Perspect Med 2:a013482). With erythroblast-induced bonepathology, attendant pathologies include low bone mineral density,osteoporosis, and bone pain (Haidar et al., 2011, Bone 48:425-432). Withiron overload, attendant pathologies include hepcidin suppression andhyperabsorption of dietary iron (Musallam et al., 2012, Blood Rev26(Suppl 1):S16-S19), multiple endocrinopathies and liverfibrosis/cirrhosis (Galanello et al., 2010, Orphanet J Rare Dis 5:11),and iron-overload cardiomyopathy (Lekawanvij it et al., 2009, Can JCardiol 25:213-218).

The most common causes of ineffective erythropoiesis are the thalassemiasyndromes, hereditary hemoglobinopathies in which imbalances in theproduction of intact alpha- and beta-hemoglobin chains lead to increasedapoptosis during erythroblast maturation (Schrier, 2002, Curr OpinHematol 9:123-126). Thalassemias are collectively among the mostfrequent genetic disorders worldwide, with changing epidemiologicpatterns predicted to contribute to a growing public health problem inboth the U.S. and globally (Vichinsky, 2005, Ann NY Acad Sci1054:18-24). Thalassemia syndromes are named according to theirseverity. Thus, α-thalassemias include α-thalassemia minor (also knownas α-thalassemia trait; two affected α-globin genes), hemoglobin Hdisease (three affected α-globin genes), and α-thalassemia major (alsoknown as hydrops fetalis; four affected α-globin genes). β-Thalassemiasinclude β-thalassemia minor (also known as β-thalassemia trait; oneaffected β-globin gene), β-thalassemia intermedia (two affected β-globingenes), hemoglobin E thalassemia (two affected β-globin genes), andβ-thalassemia major (also known as Cooley's anemia; two affectedβ-globin genes resulting in a complete absence of β-globin protein).β-Thalassemia impacts multiple organs, is associated with considerablemorbidity and mortality, and currently requires life-long care. Althoughlife expectancy in patients with β-thalassemia has increased in recentyears due to use of regular blood transfusions in combination with ironchelation, iron overload resulting both from transfusions and fromexcessive gastrointestinal absorption of iron can cause seriouscomplications such as heart disease, thrombosis, hypogonadism,hypothyroidism, diabetes, osteoporosis, and osteopenia (Rund et al,2005, N Engl J Med 353:1135-1146).

TWSG polypeptides, optionally combined with an EPO receptor activator,can be used for treating disorders of ineffective erythropoiesis besidesthalassemia syndromes. Such disorders include siderblastic anemia(inherited or acquired); dyserythropoietic anemia (Types I and II);sickle cell anemia; hereditary spherocytosis; pyruvate kinasedeficiency; megaloblastic anemias, potentially caused by conditions suchas folate deficiency (due to congenital diseases, decreased intake, orincreased requirements), cobalamin deficiency (due to congenitaldiseases, pernicious anemia, impaired absorption, pancreaticinsufficiency, or decreased intake), certain drugs, or unexplainedcauses (congenital dyserythropoietic anema, refractory megaloblasticanemia, or erythroleukemia); myelophthisic anemias, includingmyelofibrosis (myeloid metaplasia) and myelophthisis; congenitalerythropoietic porphyria; and lead poisoning.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished. In either case, prevention or treatment may be discerned inthe diagnosis provided by a physician or other health care provider andthe intended result of administration of the therapeutic agent.

In certain embodiments, agents of the invention may be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic agents such that thesecond agent is administered while the previously administeredtherapeutic agent is still effective in the body (e.g., the two agentsare simultaneously effective in the subject, which may includesynergistic effects of the two agents). For example, the differenttherapeutic agents can be administered either in the same formulation orin separate formulations, either concomitantly or sequentially. Incertain embodiments, the different therapeutic agents can beadministered within about one hour, about 12 hours, about 24 hours,about 36 hours, about 48 hours, about 72 hours, or about a week of oneanother. Thus, a subject who receives such treatment can benefit from acombined effect of different therapeutic agents.

As shown herein, TWSG polypeptides, optionally combined with an EPOreceptor activator, may be used to increase red blood cell, hemoglobinor reticulocyte levels in healthy individuals, and such TWSGpolypeptides may be used in selected patient populations. Examples ofappropriate patient populations include those with undesirably low redblood cell or hemoglobin levels, such as patients having an anemia, andthose that are at risk for developing undesirably low red blood cell orhemoglobin levels, such as those patients that are about to undergomajor surgery or other procedures that may result in substantial bloodloss. In some embodiments, a patient with adequate red blood cell levelsis treated with a TWSG polypeptide to increase red blood cell levels,and then blood is drawn and stored for later use in transfusions.

TWSG polypeptides, optionally combined with an EPO receptor activator,disclosed herein may be used to increase red blood cell levels inpatients having an anemia. When observing hemoglobin levels in humans, alevel of less than normal for the appropriate age and gender categorymay be indicative of anemia, although individual variations are takeninto account. For example, a hemoglobin level of 12 g/dL is generallyconsidered the lower limit of normal in the general adult population.Potential causes include blood-loss, nutritional deficits, medicationreaction, various problems with the bone marrow and many diseases. Moreparticularly, anemia has been associated with a variety of disordersthat include, for example, chronic renal failure, myelodysplasticsyndrome, rheumatoid arthritis, bone marrow transplantation. Anemia mayalso be associated with the following conditions: solid tumors (e.g.,breast cancer, lung cancer, colon cancer); tumors of the lymphaticsystem (e.g., chronic lymphocyte leukemia, non-Hodgkins and Hodgkinslymphomas); tumors of the hematopoietic system (e.g., leukemia,myelodysplastic syndrome, multiple myeloma); radiation therapy;chemotherapy (e.g., platinum containing regimens); inflammatory andautoimmune diseases, including, but not limited to, rheumatoidarthritis, other inflammatory arthritides, systemic lupus erythematosis(SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatorybowel disease (e.g., Crohn's disease and ulcerative colitis); acute orchronic renal disease or failure including idiopathic or congenitalconditions; acute or chronic liver disease; acute or chronic bleeding;situations where transfusion of red blood cells is not possible due topatient allo- or auto-antibodies and/or for religious reasons (e.g.,some Jehovah's Witnesses); infections (e.g., malaria, osteomyelitis);hemoglobinopathies, including, for example, sickle cell disease,thalassemias; drug use or abuse, e.g. alcohol misuse; pediatric patientswith anemia from any cause to avoid transfusion; and elderly patients orpatients with underlying cardiopulmonary disease with anemia who cannotreceive transfusions due to concerns about circulatory overload.

Myelodysplastic syndrome (MDS) is a diverse collection of hematologicalconditions characterized by ineffective production of myeloid bloodcells and risk of transformation to acute mylogenous leukemia. In MDSpatients, blood stem cells do not mature into healthy red blood cells,white blood cells, or platelets. MDS disorders include, for example,refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, refractory cytopenia with multilineagedysplasia, and myelodysplastic syndrome associated with an isolated 5qchromosome abnormality. As these disorders manifest as irreversibledefects in both quantity and quality of hematopoietic cells, most. MDSpatients are afflicted with chronic anemia. Therefore, MDS patientseventually require blood transfusions and/or treatment with growthfactors (e.g., erythropoietin or G-CSF) to increase red blood celllevels. However, many MDS patients develop side-effect due to frequencyof such therapies. For example, patients who receive frequent red bloodcell transfusion can have tissue and organ damage from the buildup ofextra iron. TWSG polypeptides disclosed herein may be used to treatpatients having MDS. In certain embodiments, patients suffering from MDSmay be treated using a combination of a TWSG polypeptide in combinationwith an EPO receptor activator. In other embodiments, patient sufferingfrom MDS may be treated using a combination of a TWSG polypeptide andone or more additional therapeutic agents for treating MDS including,for example, thalidomide, lenalidomide, azacitadine, decitabine,erythropoietins, deferoxamine, antihymocyte globulin, filgrastrim(G-CSF) and an erythropoietin signaling pathway agonist.

TWSG polypeptides, optionally combined with an EPO receptor activator,would be appropriate for treating anemias of hypoproliferative bonemarrow, which are typically associated with little change in red bloodcell (RBC) morphology. Hypoproliferative anemias include: 1) anemia ofchronic disease, 2) anemia of kidney disease, and 3) anemia associatedwith hypometabolic states. In each of these types, endogenouserythropoietin levels are inappropriately low for the degree of anemiaobserved. Other hypoproliferative anemias include: 4) early-stageiron-deficient anemia, and 5) anemia caused by damage to the bonemarrow. In these types, endogenous erythropoietin levels areappropriately elevated for the degree of anemia observed.

The most common type is anemia of chronic disease, which encompassesinflammation, infection, tissue injury, and conditions such as cancer,and is distinguished by both low erythropoietin levels and an inadequateresponse to erythropoietin in the bone marrow (Adamson, 2008, Harrison'sPrinciples of Internal Medicine, 17th ed.; McGraw Hill, New York, pp628-634). Many factors can contribute to cancer-related anemia. Some areassociated with the disease process itself and the generation ofinflamatory cytokines such as interleukin-1, interferon-gamma, and tumornecrosis factor (Bron et al., 2001, Semin Oncol 28(Suppl 8):1-6). Amongits effects, inflammation induces the key iron-regulatory peptidehepcidin, thereby inhibiting iron export from macrophages and generallylimiting iron availability for erythropoiesis (Ganz, 2007, J Am SocNephrol 18:394-400). Blood loss through various routes can alsocontribute to cancer-related anemia. The prevalence of anemia due tocancer progression varies with cancer type, ranging from 5% in prostatecancer up to 90% in multiple myeloma. Cancer-related anemia has profoundconsequences for patients, including fatigue and reduced quality oflife, reduced treatment efficacy, and increased mortality.

Chronic kidney disease is associated with hypoproliferative anemia thatvaries in severity with the degree of renal impairment. Such anemia isprimarily due to inadequate production of erythropoietin and reducedsurvival of red blood cells. Chronic kidney disease usually proceedsgradually over a period of years or decades to end-stage (Stage-5)disease, at which point dialysis or kidney transplantation is requiredfor patient survival. Anemia often develops early in this process andworsens as disease progresses. The clinical consequences of anemia ofkidney disease are well-documented and include development of leftventricular hypertrophy, impaired cognitive function, reduced quality oflife, and altered immune function (Levin et al., 1999, Am J Kidney Dis27:347-354; Nissenson, 1992, Am J Kidney Dis 20(Suppl 1):21-24; Revickiet al., 1995, Am J Kidney Dis 25:548-554; Gafter et al., 1994, KidneyInt 45:224-231). A TWSG polypeptide, optionally combined with an EPOreceptor activator, can be used to treat anemia of kidney disease.

Many conditions resulting in a hypometabolic rate can produce amild-to-moderate hypoproliferative anemia. Among such conditions areendocrine deficiency states. For example, anemia can occur in Addison'sdisease, hypothyroidism, hyperparathyroidism, or males who are castratedor treated with estrogen. Mild-to-moderate anemia can also occur withreduced dietary intake of protein, a condition particularly prevalent inthe elderly. Finally, anemia can develop in patients with chronic liverdisease arising from nearly any, cause (Adamson, 2008, Harrison'sPrinciples of Internal Medicine, 17th ed.; McGraw Hill, New York, pp628-634).

Anemia resulting from acute blood loss of sufficient volume, such asfrom trauma or postpartum hemorrhage, is known as acute post-hemorrhagicanemia. Acute blood loss initially causes hypovolemia without anemiasince there is proportional depletion of RBCs along with other bloodconstituents. However, hypovolemia will rapidly trigger physiologicmechanisms that shift fluid from the extravascular to the vascularcompartment, which results in hemodilution and anemia. If chronic, bloodloss gradually depletes body iron stores and eventually leads to irondeficiency. As demonstrated by the Applicants in a mouse model (seeExample below), a TWSG polypeptide, optionally combined with an EPOreceptor activator, can be used to speed recovery from anemia of acuteblood loss.

Iron-deficiency anemia is the final stage in a graded progression ofincreasing iron deficiency which includes negative iron balance andiron-deficient erythropoiesis as intermediate stages. Iron deficiencycan result from increased iron demand, decreased iron intake, orincreased iron loss, as exemplified in conditions such as pregnancy,inadequate diet, intestinal malabsorption, acute or chronicinflammation, and acute or chronic blood loss. With mild-to-moderateanemia of this type, the bone marrow remains hypoproliferative, and RBCmorphology is largely normal; however, even mild anemia can result insome microcytic hypochromic RBCs, and the transition to severeiron-deficient anemia is accompanied by hyperproliferation of the bonemarrow and increasingly prevalent microcytic and hypochromic RBCs(Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.;McGraw Hill, New York, pp 628-634). Appropriate therapy foriron-deficiency anemia depends on its cause and severity, with oral ironpreparations, parenteral iron formulations, and RBC transfusion as majorconventional options. A TWSG polypeptide, optionally combined with anEPO receptor activator, could be used to treat chronic iron-deficiencyanemias alone or in combination with conventional therapeuticapproaches, particularly to treat anemias of multifactorial origin.

Hypoproliferative anemias can result from primary dysfunction or failureof the bone marrow, instead of dysfunction secondary to inflammation,infection, or cancer progression. Prominent examples would bemyelosuppression caused by cancer chemotherapeutic drugs or cancerradiation therapy. A broad review of clinical trials found that mildanemia can occur in 100% of patients after chemotherapy, while moresevere anemia can occur in up to 80% of such patients (Groopman et al.,1999, J Natl Cancer Inst 91:1616-1634). Myelosuppressive drugsinclude: 1) alkylating agents such as nitrogen mustards (e.g.,melphalan) and nitrosoureas (e.g., streptozocin); 2) antimetabolitessuch as folic acid antagonists (e.g., methotrexate), purine analogs(e.g., thioguanine), and pyrimidine analogs (e.g., gemcitabine); 3)cytotoxic antibiotics such as anthracyclines (e.g., doxorubicin); 4)kinase inhibitors (e.g., gefitinib); 5) mitotic inhibitors such astaxanes (e.g., paclitaxel) and vinca alkaloids (e.g., vinorelbine); 6)monoclonal antibodies (e.g., rituximab); and 7) topoisomerase inhibitors(e.g., topotecan and etoposide). A TWSG polypeptide, optionally combinedwith an EPO receptor activator, can be used to treat anemia caused bychemotherapeutic agents and/or radiation therapy.

In patients who receive frequent transfusions of whole blood or redblood cells, normal mechanisms of iron homeostasis can be overwhelmed,eventually leading to toxic and potentially fatal accumulation of ironin vital tissues such as heart, liver, and endocrine glands. Regular redblood cell transfusions require exposure to various donor units of bloodand hence a higher risk of alloimmunization. Difficulties with vascularaccess, availability of and compliance with iron chelation, and highcost are some of the reasons why it can be beneficial to limit thenumber of red blood cell transfusions. In some embodiments, a TWSGpolypeptide may administered to a patient that has been administered oneor more blood cell transfusions (whole or red blood cell transfusions).In some embodiments, the disclosure relates to methods using a TWSGpolypeptide to treat, prevent, or reduce the progression rate and/orseverity anemia or anemia-related disorder in patient that is blood celltransfusion-dependent. In certain aspects, a TWSG polypeptide may beused to decrease (reduce) blood cell transfusion burden in a patientwith anemia or anemia-related disorder. For example, a TWSG polypeptidemay be used to decrease blood cell transfusion by greater than about30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% for 4 to 8 weeks relative tothe equal time prior to the start of the TWSG polypeptide treatment in apatient with anemia or an anemia-related disorder. In some embodiments,a TWSG polypeptide may be used to decrease blood cell transfusion bygreater than about 50% for 4 to 8 weeks relative to the equal time priorto the start of the TWSG polypeptide treatment in a patient with anemiaor an anemia-related disorder. In certain aspects, a TWSG polypeptidemay be used to decrease iron overload in a patient with anemia oranemia-related disorder. For example, a TWSG polypeptide may be used todecrease iron overload in an organ and/or tissue in a patient withanemia or an anemia-related disorder. In some embodiments, a TWSGpolypeptide may be used to decrease iron overload in the spleen of apatient with anemia or an anemia-related disorder. In some embodiments,a TWSG polypeptide may be used to decrease iron overload in the liver ofa patient with anemia or an anemia-related disorder. In someembodiments, a TWSG polypeptide may be used to decrease iron overload inthe heart of a patient with anemia or an anemia-related disorder.

TWSG polypeptides, optionally combined with an EPO receptor activator,would also be appropriate for treating anemias of disordered RBCmaturation, which are characterized in part by undersized (microcytic),oversized (macrocytic), misshapen, or abnormally colored (hypochromic)RBCs.

In certain embodiments, TWSG polypeptides may be used in combination(e.g., administered at the same time or different times, but generallyin such a manner as to achieve overlapping pharmacologic effects) withsupportive therapies for ineffective erythropoiesis. Such therapiesinclude transfusion with either red blood cells or whole blood to treatanemia. In chronic or hereditary anemias, normal mechanisms for ironhomeostasis are overwhelmed by repeated transfusions, eventually leadingto toxic and potentially fatal accumulation of iron in vital tissuessuch as heart, liver, and endocrine glands. Thus, supportive therapiesfor patients chronically afflicted with ineffective erythropoiesis alsoinclude treatment with one or more iron-chelating molecules to promoteiron excretion in the urine and/or stool and thereby prevent, orreverse, tissue iron overload (Hershko, 2006, Haematologica91:1307-1312; Cao et al, 2011, Pediatr Rep 3(2):e17). Effectiveiron-chelating agents must be able to selectively bind and neutralizeferric iron, the oxidized form of non-transferrin bound iron whichlikely accounts for most iron toxicity through catalytic production ofhydroxyl radicals and oxidation products (Esposito et al, 2003, Blood102:2670-2677). These agents are structurally diverse, but all possessoxygen or nitrogen donor atoms able to form neutralizing octahedralcoordination complexes with individual iron atoms in stoichiometries of1:1 (hexadentate agents), 2:1 (tridentate), or 3:1 (bidentate)(Kalinowski et al, 2005, Pharmacol Rev 57:547-583). Effectiveiron-chelating agents also are relatively low molecular weight (lessthan 700 daltons), with solubility in both water and lipid to enableaccess to affected tissues. Specific examples of iron-chelatingmolecules are deferoxamine, a hexadentate agent of bacterial originrequiring daily parenteral administration, and the orally activesynthetic agents deferiprone (bidentate) and deferasirox (tridentate).Combination therapy consisting of same-day administration of twoiron-chelating agents shows promise in patients unresponsive tochelation monotherapy and also in overcoming issues of poor patientcompliance with dereroxamine alone (Cao et al, 2011, Pediatr Rep3(2):e17; Galanello et al, 2010, Ann NY Acad Sci 1202:79-86).

In certain embodiments, TWSG polypeptides may be used in combinationwith hepcidin agonists for ineffective erythropoiesis. A circulatingpolypeptide produced mainly in the liver, hepcidin is considered amaster regulator of iron metabolism by virtue of its ability to inducethe degradation of ferroportin, an iron-export protein localized onabsorptive enterocytes, hepatocytes, and macrophages. Broadly speaking,hepcidin reduces availability of extracellular iron, so hepcidinagonists may be beneficial in the treatment of ineffectiveerythropoiesis (Nemeth, 2010, Adv Hematol 2010:750643). This view issupported by beneficial effects of increased hepcidin expression in amouse model of β-thalassemia (Gardenghi et al, 2010, J Clin Invest120:4466-4477).

Additionally, TWSG polypeptides may be used in combination with EPOreceptor activators to achieve an increase in red blood cells at lowerdose ranges. This may be beneficial in reducing the known off-targeteffects and risks associated with high doses of EPO receptor activators.In certain embodiments, the present invention provides methods oftreating or preventing anemia in an individual in need thereof byadministering to the individual a therapeutically effective amount of aTWSG polypeptide or a combination (or concomitant therapy) of a TWSGpolypeptide and a EPO receptor activator. These methods may be used fortherapeutic and prophylactic treatments of mammals, and particularlyhumans.

The TWSG polypeptides may be used in combination with EPO receptoractivators to reduce the required dose of these activators in patientsthat are susceptible to adverse effects of EPO. The primary adverseeffects of EPO are an excessive increase in the hematocrit or hemoglobinlevels and polycythemia. Elevated hematocrit levels can lead tohypertension (more particularly aggravation of hypertension) andvascular thrombosis. Other adverse effects of EPO which have beenreported, some of which related to hypertension, are headaches,influenza-like syndrome, obstruction of shunts, myocardial infarctionsand cerebral convulsions due to thrombosis, hypertensive encephalopathy,and red cell blood cell applasia (Singibarti, (1994) J. Clin Investig72(suppl 6), S36-S43; Horl et al. (2000) Nephrol Dial Transplant15(suppl 4), 51-56; Delanty et al. (1997) Neurology 49, 686-689; Bunn(2002) N Engl J Med 346(7), 522-523).

Patients may be treated with a dosing regimen intended to restore thepatient to a target hemoglobin level, usually between about 10 g/dl andabout 12.5 g/dl, and typically about 11.0 g/dl (see also Jacobs et al.(2000) Nephrol Dial Transplant 15, 15-19), although lower target levelsmay cause fewer cardiovascular side effects. Alternatively, hematocritlevels (percentage of the volume of a blood sample occupied by thecells) can be used as a measure for the condition of red blood cells.Hematocrit levels for healthy individuals range from 41 to 51% for adultmales and from 35 to 45% for adult females. Target hematocrit levels areusually around 30-33%. Moreover, hemoglobin/hematocrit levels vary fromperson to person. Thus, optimally, the target hemoglobin/hematocritlevel can be individualized for each patient.

In certain embodiments, the present invention provides methods formanaging a patient that has been treated with, or is a candidate to betreated with, a TWSG polypeptide by measuring one or more hematologicparameters in the patient. The hematologic parameters may be used toevaluate appropriate dosing for a patient who is a candidate to betreated with a TWSG polypeptide, to monitor the hematologic parametersduring treatment with a TWSG polypeptide, to evaluate whether to adjustthe dosage during treatment with a TWSG polypeptide, and/or to evaluatean appropriate maintenance dose of a TWSG polypeptide. If one or more ofthe hematologic parameters are outside the normal level, dosing with aTWSG polypeptide may be reduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with themethods provided herein include, for example, red blood cell levels,blood pressure, iron stores, and other agents found in bodily fluidsthat correlate with increased red blood cell levels, using artrecognized methods. Such parameters may be determined using a bloodsample from a patient. Increases in red blood cell levels, hemoglobinlevels, and/or hematocrit levels may cause increases in blood pressure.

In some embodiments, if one or more hematologic parameters are outsidethe normal range, or on the high side of normal, in a patient who is acandidate to be treated with a TWSG polypeptide then onset ofadministration of the TWSG polypeptide may be delayed until thehematologic parameters have returned to a normal or acceptable leveleither naturally or via therapeutic intervention. For example, if acandidate patient is hypertensive or prehypertensive, then the patientmay be treated with a blood pressure lowering agent in order to reducethe patient's blood pressure. Any blood pressure lowering agentappropriate for the individual patient's condition may be usedincluding, for example, diuretics, adrenergic inhibitors (includingalpha blockers and beta blockers), vasodilators, calcium channelblockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensinII receptor blockers. Blood pressure may alternatively be treated usinga diet and exercise regimen. Similarly, if a candidate patient has ironstores that are lower than normal, or on the low side of normal, thenthe patient may be treated with an appropriate regimen of diet and/oriron supplements until the patient's iron stores have returned to anormal or acceptable level. For patients having higher than normal redblood cell levels and/or hemoglobin levels, then administration of theTWSG polypeptide may be delayed until the levels have returned to anormal or acceptable level.

In certain embodiments, if one or more hematologic parameters areoutside the normal range, or on the high side of normal, in a patientwho is a candidate to be treated with a TWSG polypeptide then the onsetof administration may be not be delayed. However, the dosage amount orfrequency of dosing of the TWSG polypeptide may be set at an amount thatwould reduce the risk of an unacceptable increase in the hematologicparameters arising upon administration of the TWSG polypeptide.Alternatively, a therapeutic regimen may be developed for the patientthat combines a TWSG polypeptide with a therapeutic agent that addressesthe undesirable level of the hematologic parameter. For example, if thepatient has elevated blood pressure, then a therapeutic regimeninvolving administration of a TWSG polypeptide and a blood pressurelowering agent may be designed. For a patient having lower than desirediron stores, a therapeutic regimen of a TWSG polypeptide and ironsupplementation may be developed.

In some embodiments, baseline parameter(s) for one or more hematologicparameters may be established for a patient who is a candidate to betreated with a TWSG polypeptide and an appropriate dosing regimenestablish for that patient based on the baseline value(s).Alternatively, established baseline parameters based on a patient'smedical history could be used to inform an appropriate TWSG polypeptidedosing regimen for a patient. For example, if a healthy patient has anestablished baseline blood pressure reading that is above the definednormal range it may not be necessary to bring the patient's bloodpressure into the range that is considered normal for the generalpopulation prior to treatment with the TWSG polypeptide. A patient'sbaseline values for one or more hematologic parameters prior totreatment with a TWSG polypeptide may also be used as the relevantcomparative values for monitoring any changes to the hematologicparameters during treatment with the TWSG polypeptide.

In certain embodiments, one or more hematologic parameters are measuredin patients who are being treated with a TWSG polypeptide. Thehematologic parameters may be used to monitor the patient duringtreatment and permit adjustment or termination of the dosing with theTWSG polypeptide or additional dosing with another therapeutic agent.For example, if administration of a TWSG polypeptide results in anincrease in blood pressure, red blood cell level, or hemoglobin level,or a reduction in iron stores, then the dose of the TWSG polypeptide maybe reduced in amount or frequency in order to decrease the effects ofthe TWSG polypeptide on the one or more hematologic parameters. Ifadministration of a TWSG polypeptide results in a change in one or morehematologic parameters that is adverse to the patient, then the dosingof the TWSG polypeptide may be terminated either temporarily, until thehematologic parameter(s) return to an acceptable level, or permanently.Similarly, if one or more hematologic parameters are not brought withinan acceptable range after reducing the dose or frequency ofadministration of the TWSG polypeptide then the dosing may beterminated. As an alternative, or in addition to, reducing orterminating the dosing with the TWSG polypeptide, the patient may bedosed with an additional therapeutic agent that addresses theundesirable level in the hematologic parameter(s), such as, for example,a blood pressure lowering agent or an iron supplement. For example, if apatient being treated with a TWSG polypeptide has elevated bloodpressure, then dosing with the TWSG polypeptide may continue at the samelevel and a blood pressure lowering agent is added to the treatmentregimen, dosing with the TWSG polypeptide may be reduce (e.g., in amountand/or frequency) and a blood pressure lowering agent is added to thetreatment regimen, or dosing with the TWSG polypeptide may be terminatedand the patient may be treated with a blood pressure lowering agent.

6. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., TWSG polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, a TWSG polypeptide can be administered alone or asa component of a pharmaceutical formulation (therapeutic composition).The subject compounds may be formulated for administration in anyconvenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition systemically, or locally, e.g., using animplant or device. When administered, the therapeutic composition foruse in this invention may be in any physiologically acceptable form,such as in a pyrogen-free composition. Therapeutically useful agentsother than the TWSG polypeptides which may also optionally be includedin the composition as described above, may be administeredsimultaneously or sequentially with the subject compounds (e.g., TWSGpolypeptides) in the methods of the invention.

Typically, compounds will be administered parenterally. Pharmaceuticalcompositions suitable for parenteral administration may comprise one ormore TWSG polypeptides in combination with one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions justprior to use, which may contain antioxidants, buffers, bacteriostats,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents. Examples ofsuitable aqueous and nonaqueous carriers which may be employed in thepharmaceutical compositions of the invention include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site (e.g., bone marrow). In certainembodiments, compositions of the present invention may include a matrixcapable of delivering one or more therapeutic compounds (e.g., TWSGpolypeptides) to a target tissue site (e.g., bone marrow), providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the TWSG polypeptides. Such matrices may be formed of materialssuitable for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., TWSG polypeptides). Thevarious factors include, but are not limited to, the patient's red bloodcell count, hemoglobin level or other diagnostic assessments, thedesired target red blood cell count, the patient's age, sex, and diet,the severity of any disease that may be contributing to a depressed redblood cell level, time of administration, and other clinical factors.The addition of other known growth factors to the final composition mayalso affect the dosage. Progress can be monitored by periodic assessmentof red blood cell and hemoglobin levels, as well as assessments ofreticulocyte levels and other indicators of the hematopoietic process.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of TWSG polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the TWSGpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of TWSG polynucleotide sequences can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Preferred for therapeutic delivery of TWSGpolynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus suchas a retrovirus. The retroviral vector may be a derivative of a murineor avian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous. Sarcoma Virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the TWSGpolynucleotide.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for TWSG polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane-vesicles which are useful asdelivery vehicles in vitro and in vivo. RNA, DNA and intact virions canbe encapsulated within the aqueous interior and be delivered to cells ina biologically active form (see e.g., Fraley, et al., Trends Biochem.Sci., 6:77, 1981). Methods for efficient gene transfer using a liposomevehicle, are known in the art, see e.g., Mannino, et al., Biotechniques,6:682, 1988. The composition of the liposome is usually a combination ofphospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of Human TWSG-Fc Fusion Protein

Applicants constructed a soluble TWSG fusion protein (TWSG-Fc) in whichfull-length human TWSG (FIG. 1, SEQ ID NO: 8) was attached at itsC-terminus to a human IgG₁ Fc domain (SEQ ID NO: 3) with a minimallinker. TWSG-Fc (FIG. 2, SEQ ID NO: 9) was initially expressed bytransient transfection in COS cells, as were N-glycosylation variants ofTWSG-Fc (see below). In brief, COS cells (ATCC®) were transfectedovernight with plasmid encoding TWSG-Fc using FuGENE® 6 transfectionreagent (Promega). The next day, cells were washed withphosphate-buffered saline, and serum-free medium was added. Afterincubation for 72 h, the COS-conditioned medium was harvested, filtered,and loaded on a MabSelect SuRe column (GE Healthcare, UK). Fusionprotein was eluted with 0.1 M glycine (pH 3.0), and the eluted fractionswere immediately neutralized by addition 1 M Tris (pH 8.0) in a 1:10ratio. Protein was quantitated using a NanoDrop™ spectrophotometer(Thermo Fisher Scientific, Waltham, Mass.).

CHO cells were transfected by standard methods with plasmid encodingTWSG-Fc and containing a ubiquitous chromatin opening element (UCOE) tofacilitate protein expression. See, e.g., Cytotechnology (2002)38:43-46. Pools were selected in methotrexate (MTX) at concentrations of10 nM, 20 nM, and 50 nM. The 50 nM MTX pool yielded the highestexpression level, so a dilution clone was obtained from this pool andadapted to serum-free suspension growth to generate conditioned mediafor purification.

Three different leader sequences may be used:

(i) the leader sequence for Honey bee mellitin (HBML): (SEQ ID NO: 10)MKFLVNVALVFMVVYISYIYA (ii) the leader sequence for Tissue plasminogenactivator (TPA): (SEQ ID NO: 11) MDAMKRGLCCVLLLCGAVFVSP(iii) the leader sequence for Native human TWSG: (SEQ ID NO: 12)MKLHYVAVLTLAILMFLTWLPESLS

The selected form of TWSG-Fc uses the TPA leader, has the unprocessedamino acid sequence shown in FIG. 3 (SEQ ID NO: 13), and is encoded bythe nucleotide sequence shown in FIG. 4 (SEQ ID NO: 14). Applicants alsoenvision an alternative TWSG-hFc sequence with or without TPA leadercomprising a different hFc domain (for example, SEQ ID NOs: 4, 5, 6, or7, or a chimeric Fc domain from different IgG origins, such as chimericG2/G4 constant regions) attached to at least one end of the C-terminusand N-terminus of human TWSG by a minimal linker.

Purification of Fusion Protein Derived from CHO Cells

Human TWSG-Fc expressed in CHO cells was purified as follows forsubsequent characterization by surface plasmon resonance and reportergene assays. Conditioned medium containing hTWSG-hFc was concentrated,filtered, and loaded on a MAb SelectSuRe column previously equilibratedwith PBS. Resin was then washed with PBS, and the protein was elutedwith 0.1M glycine pH 3.5. Fractions containing protein were neutralizedwith 5% (v/v) 1M Tris pH 8.0. The elution pool was loaded on a QSepharose FF 10 mL column (GE Healthcare) previously equilibrated withbuffers A (50 mM Tris pH 8.0) and B (50 mM Tris, 1M NaCl pH 8.0). A washwas performed at 10% B (100 mM NaCl), followed by elution at 20% B (200mM NaCl). Protein was further processed over HiLoad™ 26/60 Superdex (GEHealthcare) equilibrated in PBS containing 50 mM arginine (pH 7.22).Fractions were evaluated by analytical size-exclusion chromatography,and those containing over 90% monomer were pooled, concentrated, andcharacterized. Purity of samples was evaluated by analyticalsize-exclusion chromatography and SDS-PAGE with Coomassie staining.Analysis indicated that the mature protein has an N-terminal sequence ofCNKAL (SEQ ID NO: 15).

Example 2. Ligand Binding to Murine TWSG and Human TWSG-Fc

Previous studies have determined that TWSG, or its nonmammalian homologTsg, binds with high affinity to BMP2, BMP4, and BMP7 (Oelgeschläger etal., 2000, Nature 405:757-763; Scott et al., 2001, Nature 410:475-478;Chang et al., 2001, Nature 410:483-487). Since these studies have notsystematically evaluated TWSG (or Tsg) binding to other TGFβ superfamilyligands, Applicants used surface plasmon resonance to investigate andcharacterize such binding. In an initial qualitative screen, recombinantmurine TWSG (mTWSG; R&D Systems, Minneapolis, Minn.) was covalentlyimmobilized on a BIACORE™ CMS chip, and more than 30 ligands generatedin-house or obtained from R&D Systems were injected individually overthe captured mTWSG to characterize their degree of binding at roomtemperature. Based on the results of this screen, Applicants subjectedselected ligands to quantitative characterization of binding to humanTWSG fusion protein at physiologic temperature. For this analysis,TWSG-Fc was expressed in CHO cells, purified as described in Example 1,captured on a BIACORE™ chip with anti-Fc antibody, and tested by surfaceplasmon resonance with the following ligands at 37° C.

Ligand* k_(a) (10⁶ M⁻¹s⁻¹) k_(d) (10⁻³ s⁻¹) K_(D) (nM) BMP4  1.9  1.40.23 BMP6  8.2  1.9 0.23 BMP2  7.4  2.2 0.30 BMP7  4.8  1.6 0.33 BMP966.0 25.0 4.4  BMP10 — — No binding *All ligands (R&D Systems) werehuman.

As shown in the table above, human TWSG-Fc can bind with sub-nanomolaraffinity to four TGFβ superfamily ligands (BMP2, BMP4, BMP6, BMP7).Although TWSG-Fc exhibited low nanomolar affinity for BMP9 atequilibrium, the off-rate (k_(d)) for BMP9 was at least an order ofmagnitude faster than for any of the other ligands shown, correspondingto a mean residence time for the ligand-receptor complex ofapproximately 1 minute. This relatively short residence time likelyexplains the inability of TWSG-Fc fusion protein to inhibit BMP9signaling in a cell-based assay (see Example 3). Even when expressedstably in CHO cells, TWSG-Fc displayed no binding to the closely relatedligand BMP10, as determined by surface plasmon resonance.

The affinity of murine TWSG for BMP2 and BMP4 has been reported to varywith its glycosylation status (Billington Jr. et al., 2011, FrontPhysiol 2:59). Therefore, Applicants generated several glycosylationvariants of hTWSG-hFc by mutating existing sites (FIG. 3) of N-linkedglycosylation in the hTWSG sequence, both individually and incombination, and expressing the constructs transiently in COS cells.Unlike reports of murine variants, ligand binding properties of thesehuman glycosylation variants as determined by surface plasmon resonanceand reporter gene assay were found to be similar to that of wild-typehuman TWSG-Fc.

Example 3. Inhibition of Ligand Signaling by TWSG-Fc in Cell-BasedAssays

Reporter gene assays were used to determine the ability of human TWSG-Fcto inhibit signaling by BMP2, BMP4, BMP6, BMP7, BMP9, and BMP10. Theseassays are based on human glioblastoma (T98G) or hepatocellularcarcinoma (HepG2) cell lines transfected with a pGL3 BRE (BMP responseelement) reporter plasmid (Korchynskyi et al., 2002, J Biol Chem 277:4883-4891) as well as a Renilla reporter plasmid (pRLCMV) to control fortransfection efficiency. BMP response elements from the Id1 promoter arepresent in the promoter of the pGL3 BRE reporter plasmid, so this vectoris of general use for factors signaling through Smad1 and Smad5.

On the first day of the assay, T98G cells (ATCC®: CRL_1690™) or HepG2cells (ATCC®: HB-8065™) were distributed in 48-well plates at 8.5×10⁴cells per well or 12.5×10⁴ cells per well, respectively. On the secondday, a solution containing 10 μg pGL3 BRE, 100 ng pRLCMV, 30 μl FugeneHD (Roche Applied Science, DE), and 970 μl OptiMEM™ (Invitrogen) waspreincubated for 30 min, then added to assay buffer consisting of eitherEagle's minimum essential medium, ATCC® (T98G), or McCoy's 5A medium,Life Technologies® (HepG2), supplemented with 0.1% BSA. The mixture wasapplied to the plated cells (500 μl/well) for incubation overnight at37° C.

On the third day, medium was removed, and cells were incubated overnightat 37° C. with a mixture of ligands and inhibitors prepared as describedbelow. TWSG-Fc was serially diluted in 200 μl volumes of assay bufferusing a 48-well plate. An equal volume of assay buffer containing thetest ligand was added to obtain a final ligand concentration equal tothe EC₅₀ determined previously. Human BMP2, BMP4, BMP6, BMP7, BMP9, andBMP10 were obtained from R&D Systems. Test solutions were incubated at37° C. for 30 minutes, then 250 μl of the mixture was added to allwells. Each concentration of test article was determined in duplicate.After incubation with test solutions overnight, cells were rinsed withphosphate-buffered saline, then lysed with passive lysis buffer (PromegaE1941) and stored overnight at −70° C. On the fourth and final day,plates were warmed to room temperature with gentle shaking. Cell lysateswere transferred in duplicate to a chemiluminescence plate (96-well) andanalyzed in a luminometer with reagents from a Dual-Luciferase ReporterAssay system (Promega El 980) to determine normalized luciferaseactivity.

These assays were used to evaluate the ability of TWSG-Fc to inhibitcell signaling mediated by BMPs that applicants identified by surfaceplasmon resonance as high-affinity binders. TWSG-Fc used in these assayswas expressed in CHO cells and purified as described above.

IC₅₀ Ligand ng/mL nM BMP7  143  1.5 BMP4  208  2.2 BMP6  351  3.7 BMP22960 31   BMP9 ND (>3000) ND (>31) BMP10 ND (>3000) ND (>31) Values arethe means of two separate experiments. ND, not determined due toweakness of inhibition.TWSG-hFc displayed approximately equal potency as an inhibitor of BMP4,BMP6, and BMP7, with IC₅₀ values in the low nanomolar range, whereas itwas an order of magnitude less potent at inhibiting BMP2. As expectedfrom surface plasmon resonance analysis, TWSG-Fc did not appreciablyinhibit signaling by BMP9 or BMP10.

Example 4. Stimulatory Effect of TWSG-Fc on Iron and Erythrocyte Levelsin Mice

Inflammation associated with chronic diseases, autoimmune diseases, andinfection can promote anemia, for which there are limited treatmentoptions (Roy, 2010, Hematology Am Soc Hematol Educ Program, 2010:276-80;Kwaan, 2011, Infect Disord Drug Targets 11:40-44). As demonstrated by atransgenic mouse model (Roy et al., 2007, Blood 109:4038-4044), elevatedhepcidin production is now considered the primary cause of anemia ofinflammation (Roy, 2010, Hematology Am Soc Hematol Educ Program,2010:276-80; Ganz et al., 2012, Biochim Biophys Acta 1823:1434-1443).BMP6, in turn, has been identified as a key endogenous regulator ofhepcidin expression (Camaschella, 2009, Nat Genet 41:386-388; Meynard etal., 2009, Nat Genet 41:478-481; Andriopoulos et al., 2009, Nat Genet41:482-487).

Given the ability of human TWSG-Fc to inhibit signaling mediated by BMP6in particular, Applicants investigated the effect of human TWSG-Fc oniron levels and red blood cell indices in wild-type mice. Eight-week-oldC57BL/6 mice (n=5 per group) were treated intraperitoneally with TWSG-Fc(10 mg/kg) or vehicle (phosphate-buffered saline containing 50 mMarginine) either daily for 1 week or three times per wk for 4 week.Compared with vehicle, treatment with TWSG-Fc for 1 week significantlyincreased serum iron by 12% (n=5 per group) (FIG. 5). Four weekstreatment with TWSG-Fc markedly increased iron levels in spleen (FIG. 6)but not other tissues, indicative of upregulated erythropoiesis inspleen. As shown in FIG. 6, spleen sections from wild-type mice treatedwith TWSG-Fc for 4 weeks contained numerous deposits of iron reactionproduct spread across regions of active erythropoiesis known as red pulp(right panel, showing the Prussian blue staining under microscope).Little or no staining was present in spleen sections fromvehicle-treated mice (FIG. 6, left panel). Consistent with this finding,treatment with TWSG-Fc for 4 weeks significantly increased red cellcount and hemoglobin concentration (FIG. 7). As shown in FIG. 7,compared to vehicle, treatment of wild-type mice with TWSG-Fc for 4weeks increased RBC count by 15% (FIG. 7A) and hemoglobin concentrationby 12% (FIG. 7B). In these same mice, no effects of TWSG-Fc treatmentwere observed on bone mineral density, as determined by dual energyX-ray absorptiometry, or fluid mass, fat mass, or lean body mass, asdetermined by nuclear magnetic resonance. This effect profile contrastswith that of ALK3-Fc fusion proteins, which bind with highest affinityto BMP2 and BMP4 and markedly stimulate bone growth in vivo (U.S. Pat.No. 8,338,377).

Taken together, these results show that TWSG-Fc exerts stimulatoryeffects on iron levels and RBC indices in vivo that are consistent withits ability to inhibit BMP6 in cell-based assays. Applicants hypothesizethat these effects of TWSG-Fc are mediated by reduced BMP6-dependenthepatic expression of hepcidin and reduced circulating hepcidinconcentrations. Thus, TWSG fusion proteins such as TWSG-Fc might beuseful for treating anemia of inflammation in patients in need thereof.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A polypeptide comprising a Twisted Gastrulation (TWSG) polypeptideand a fragment crystallizable region (Fc) polypeptide.
 2. (canceled) 3.The polypeptide of claim 1, wherein TWSG polypeptide is encodable by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO:
 2. 4-5.(canceled)
 6. The polypeptide of claim 1, wherein the TWSG polypeptidecomprises an amino acid sequence at least about 80% identical to thesequence of SEQ ID NO:
 8. 7-15. (canceled)
 16. The polypeptide of claim1, wherein the Fc polypeptide comprises an amino acid sequence at leastabout 80% identical to the sequence of SEQ ID NO: 3, 4, 5, 6, or 7.17-52. (canceled)
 53. A multimeric polypeptide complex comprising thepolypeptide of claim
 1. 54-56. (canceled)
 57. A composition comprisingthe polypeptide of claim 1 and a pharmaceutically acceptable carrier.58. A composition comprising the multimeric polypeptide complex of claim53 and a pharmaceutically acceptable carrier.
 59. A polynucleotideencoding the polypeptide of claim
 1. 60. (canceled)
 61. A compositioncomprising the polynucleotide of claim 59 and a pharmaceuticallyacceptable carrier.
 62. A vector comprising at least one regulatorysequence operably linked to the polynucleotide of claim
 59. 63. A hostcell expressing the vector of claim
 62. 64. A kit comprising thepolypeptide of claim 1, and, optionally, an administration device.
 65. Anon-human animal engineered to express, or to overexpress, thepolypeptide of claim
 1. 66. A method of producing a TWSG-Fc fusionpolypeptide, comprising: i) providing a cell comprising thepolynucleotide of claim 59; and ii) culturing the cell under conditionssuitable for expression of the TWSG-Fc fusion polypeptide encoded by thepolynucleotide. 67-69. (canceled)
 70. A method of inhibiting BMPsignaling in a cell, tissue, or organ, comprising administering thepolypeptide of claim
 1. 71. A method of increasing red blood cell and/orhemoglobin levels, or reducing blood transfusion-dependence (TD), in asubject, comprising administering to the subject the polypeptide ofclaim
 1. 72. A method of increasing iron levels in a subject, comprisingadministering to the subject the polypeptide of claim
 1. 73. (canceled)74. A method of treating iron overload in a subject, comprisingadministering to the subject the polypeptide of claim
 1. 75. (canceled)76. A method of treating dysregulation of BMP signaling in a subject,comprising administering to the subject the polypeptide of claim 1.77-78. (canceled)
 79. A method of treating anemia in a subject,comprising administering to the subject the polypeptide of claim
 52. 80.A method of treating anemia in a subject, comprising administering tothe subject a fusion protein comprising a Twisted Gastrulation (TWSG)polypeptide and a fragment crystallizable region (Fc) polypeptide.
 81. Amethod of treating anemia in a subject, comprising administering to thesubject a multimeric polypeptide complex comprising a fusion proteincomprising a Twisted Gastrulation (TWSG) polypeptide and a fragmentcrystallizable region (Fc) polypeptide. 82-100. (canceled)